Method for producing linear unsaturated carboxylic acid compounds using phenylalanine ammonia-lyase

Abstract

A method for producing a chain unsaturated carboxylic acid compound having at least two carbon-carbon double bonds or a geometric isomer thereof, said method comprising a step for, from a chain unsaturated carboxylic acid compound having an amino group and a terminal carbon-carbon double bond or a geometric isomer thereof, removing the amino group in the presence of phenylalanine ammonia lyase to thereby further form a carbon-carbon double bond.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a third chain unsaturated carboxylic acid compound by introducing a carbon-carbon double bond into a second chain unsaturated carboxylic acid compound having a terminal carbon-carbon double bond in the presence of phenylalanine ammonia-lyase.The present invention also relates to a method for producing a second chain unsaturated carboxylic acid compound from a first chain unsaturated carboxylic acid compound having a terminal amino group in the presence of terminal alkene-generating enzyme BesC, and then producing a third chain unsaturated carboxylic acid compound from the second chain unsaturated carboxylic acid compound in the presence of phenylalanine ammonia-lyase.Furthermore, the present invention relates to a method for producing the third chain unsaturated carboxylic acid compound in this manner, and then producing a chain unsaturated hydrocarbon compound having carbon-carbon double bonds at both terminals from the unsaturated carboxylic acid compound in the presence of ferulic acid decarboxylase.

[0002] The present invention also relates to phenylalanine ammonia-lyase variants that can be used in these production methods, DNA encoding the variants, vectors into which the DNAs have been inserted, and host cells into which the DNAs or vectors have been introduced. Furthermore, the present invention relates to methods for producing the variants using the host cells. [Background technology]

[0003] Butadiene (1,3-butadiene) is an extremely important organic compound in the chemical industry because it is used as a raw material for various polymer compounds, such as various synthetic rubbers (butadiene rubber, styrene-butadiene rubber, acrylonitrile-butadiene rubber, etc.) and polymer resins (ABS resin, nylon 66, etc.). Furthermore, these polymer compounds made from butadiene are widely used not only in industrial products such as automobile tires, but also in everyday items such as clothing. As a result, demand for butadiene is increasing year by year, reaching 13 million tons per year and a market size of $15 billion.

[0004] Butadiene has traditionally been produced by refining the C4 fraction, a by-product of the production of ethylene and propylene from petroleum. However, due to environmental issues such as the depletion of fossil fuels such as petroleum and global warming caused by greenhouse gas emissions, there is a growing need to realize sustainable butadiene production in order to meet the ever-increasing demand for butadiene. As a countermeasure, active efforts are being made to develop methods for producing butadiene from materials derived from renewable biomass resources using enzymes.

[0005] For example, Patent Document 1 discloses a method for producing butadiene using a microorganism having an enzymatic activity capable of converting xylose as a raw material into crotyl alcohol or the like. Patent Document 2 discloses a method for producing butadiene using a microorganism having an enzymatic activity capable of converting xylose as a raw material into 2,3-butanediol. Thus, there have been many attempts to produce unsaturated hydrocarbon compounds such as butadiene using enzymes.

[0006] Furthermore, the present inventors have demonstrated that the production of 4-vinylguaiacol (4VG) by decarboxylation of ferulic acid in which ferulic acid decarboxylase (FDC) is involved (see Non-Patent Document 1) can be applied to the production of unsaturated hydrocarbon compounds such as butadiene (Patent Document 3). That is, the present inventors have discovered that butadiene can be produced from muconic acid, etc., using FDC via a decarboxylation reaction as shown in the following formula:

[0007] [ka]

[0008] However, muconic acid is expensive, which may hinder its practical application. Therefore, it is desirable to develop a method for producing unsaturated compounds with at least two carbon-carbon double bonds, such as butadiene, more inexpensively using an enzyme. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-30376 [Patent Document 2] Japanese Patent Application Laid-Open No. 2015-228804 [Patent Document 3] International Publication No. 2019-022083 [Non-patent literature]

[0010] [Non-Patent Document 1] Karl AP Payne et al., Nature, 2015, Vol. 522, No. 7557, pp. 497-501 [Non-patent document 2] J.A. Marchand et al., Nature, 2019, Vol. 567, No. 7748, pp. 420-424 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention has been made in view of the problems associated with the prior art, and an object of the present invention is to provide a method for producing an unsaturated compound having at least two carbon-carbon double bonds using an enzyme. [Means for solving the problem]

[0012] As a result of extensive research to achieve the above object, the present inventors have conceived the following reaction scheme for producing butadiene using L-lysine instead of muconic acid as a starting material. Note that L-lysine is relatively inexpensive, which makes it possible to reduce the cost of butadiene production.

[0013] [ka]

[0014] Furthermore, the present inventors have conceived the idea that the enzyme BesC disclosed in Non-Patent Document 2 can be used to produce L-allylglycine from L-lysine. This enzyme has the activity of catalyzing a reaction in which a terminal propylamino group is oxidized to cleave the carbon-carbon bond in the group and form a terminal carbon-carbon double bond. Furthermore, the present inventors have conceived the idea that FDC, which has been revealed by the present inventors as described above, can be used to produce butadiene from pentadienoate (Patent Document 3).

[0015] However, no reports have been found of an enzyme involved in the production of pentadienoic acid from L-allylglycine. Therefore, the present inventors hypothesized that phenylalanine ammonia-lyase (PAL), which is involved in the production of cinnamic acid from phenylalanine as described below, could be used for this purpose.

[0016] [ka]

[0017] In fact, when E. coli that overexpressed BesC and Arabidopsis thaliana-derived PAL (AtPAL) was cultured and the cell lysate obtained by processing the culture was mixed with E. coli that overexpressed FDC and the cell lysate obtained by processing the culture, and L-lysine was added, it was found that butadiene was produced.

[0018] Furthermore, butadiene production was also examined using PALs of different origins (PAL derived from Anabaena variabilis (AvPAL) and PAL derived from Plagiochasma appendiculatum (PaPAL)) in the same manner as described above. As a result, it was revealed that butadiene can be produced using AvPAL or PaPAL, similar to the case of AtPAL. Furthermore, the present inventors found that among these, AvPAL has a significantly higher catalytic activity for the production of pentadienoic acid.

[0019] Furthermore, various amino acid substitutions were introduced into this AvPAL, and the butadiene production ability was examined. As a result, it was found that any of the following amino acid substitutions (1) to (5) improved the amount of butadiene produced compared to the wild-type strain before the substitution, and this led to the completion of the present invention. (1) substitution of leucine at position 108 with methionine, phenylalanine, or valine; (2) Phenylalanine at position 107 is replaced with tryptophan; (3) Leucine at position 219 is replaced with isoleucine; (4) Asparagine at position 223 is replaced with isoleucine; (5) Leucine at position 104 was replaced with alanine.

[0020] Specifically, the present invention relates to a method for producing a third chain unsaturated carboxylic acid compound by introducing a carbon-carbon double bond into a second chain unsaturated carboxylic acid compound having a terminal carbon-carbon double bond in the presence of PAL. The present invention also relates to a method for producing a second chain unsaturated carboxylic acid compound from a first chain unsaturated carboxylic acid compound having a terminal amino group in the presence of BesC, and then producing a third chain unsaturated carboxylic acid compound from the second chain unsaturated carboxylic acid compound in the presence of phenylalanine ammonia lyase. Furthermore, the present invention relates to a method for producing a third chain unsaturated carboxylic acid compound from the unsaturated carboxylic acid compound thus produced, in the presence of PDC.

[0021] The present invention also relates to PAL mutants that can be used in these production methods, DNA encoding the mutants, vectors into which the DNAs have been inserted, and host cells into which the DNAs or vectors have been introduced. Furthermore, the present invention relates to methods for producing the mutants using the host cells.

[0022] More specifically, the present invention provides the following: [1] A method for producing a chain unsaturated carboxylic acid compound represented by the following formula (3) or a geometric isomer thereof, comprising a step of eliminating a first amino group from a chain unsaturated carboxylic acid compound represented by the following formula (2) or a geometric isomer thereof, the chain having a first amino group and a first carbon-carbon double bond at a terminal, in the presence of phenylalanine ammonia-lyase, to form a second carbon-carbon double bond:

[0023] [ka]

[0024] [In the above formula, (A) represents a linear hydrocarbon group having 0 to 5 carbon atoms which may be substituted, and when the carbon atom number is 2 to 5, a double bond may be formed between adjacent carbon atoms. R 1 and R 2 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. [2] The method according to [1], which comprises a step of producing a chain unsaturated carboxylic acid compound represented by the following formula (2) or a geometric isomer thereof by eliminating a second amino group and a methylene group from a chain carboxylic acid compound represented by the following formula (1) or a geometric isomer thereof, which has a first amino group and a second amino group at a terminal, in the presence of a terminal alkene synthase BesC, and producing a chain unsaturated carboxylic acid compound represented by the formula (3) or a geometric isomer thereof from the compound or the geometric isomer thereof:

[0025] [ka]

[0026] [In the above formula, (A) represents a linear hydrocarbon group having 0 to 5 carbon atoms which may be substituted, and when the carbon atom number is 2 to 5, a double bond may be formed between adjacent carbon atoms. R 1 and R 2each 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. [3] A method for producing a chain unsaturated hydrocarbon compound represented by the following formula (4) or a geometric isomer thereof, comprising the steps of producing a chain unsaturated carboxylic acid compound represented by the formula (3) or a geometric isomer thereof by the method described in [1] or [2], and eliminating a carboxyl group from the unsaturated carboxylic acid compound or the geometric isomer thereof in the presence of ferulic acid decarboxylase:

[0027] [ka]

[0028] [In the above formula, (A) represents a linear hydrocarbon group having 0 to 5 carbon atoms which may be substituted, and when the carbon atom number is 2 to 5, a double bond may be formed between adjacent carbon atoms. R 1 and R 2 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. [4] The method according to any one of [1] to [3], wherein the phenylalanine ammonia-lyase has at least one of the following characteristics (1) to (5): (1) The amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is methionine, phenylalanine, or valine. (2) The amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is tryptophan. (3) The amino acid at position 219 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is isoleucine. (4) The amino acid at position 223 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is isoleucine. (5) The amino acid at position 104 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is alanine. [5] A phenylalanine ammonia-lyase variant into which at least one of the following amino acid substitutions (1) to (5) has been introduced: (1) The amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with methionine, phenylalanine, or valine; (2) the amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with tryptophan; (3) The amino acid at position 219 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with isoleucine; (4) The amino acid at position 223 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with isoleucine; (5) The amino acid at position 104 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with alanine. [6] A DNA encoding the phenylalanine ammonia-lyase variant described in [5]. [7] A vector containing the DNA described in [6]. [8] A host cell into which the DNA described in [6] or the vector described in [7] has been introduced. [9] A method for producing a phenylalanine ammonia-lyase variant, comprising the steps of culturing the host cell according to [8] and collecting the protein expressed in the host cell.

[10] A method for producing a phenylalanine ammonia-lyase variant, comprising the step of introducing at least one of the following amino acid substitutions (1) to (5) into phenylalanine ammonia-lyase: (1) replacing the amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position with methionine, phenylalanine, or valine; (2) replacing the amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position with tryptophan; (3) replacing the amino acid at position 219 of the amino acid sequence set forth in SEQ ID NO: 2 or a corresponding position with isoleucine; (4) Substitution of the amino acid at or corresponding to position 223 of the amino acid sequence set forth in SEQ ID NO: 2 with isoleucine; (5) The amino acid at position 104 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with alanine. [Effects of the Invention]

[0029] According to the present invention, it is possible to provide a method for producing an unsaturated compound having at least two carbon-carbon double bonds using an enzyme. For example, the present invention makes it possible to produce butadiene using relatively inexpensive L-lysine as a starting material. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 is a graph showing the results of adding phenylalanine to a mixed solution of phenylalanine ammonia-lyase (AtPAL, AvPAL, or PaPAL) derived from Arabidopsis thaliana, Anabaena variabilis, or Plagiochasma appendiculatum, and ferulic acid decarboxylase, and measuring the amount of styrene produced. [Figure 2] 1 is a graph showing the results of adding allylglycine to a mixed solution of AtPAL, AvPAL, or PaPAL and ferulic acid decarboxylase, and measuring the amount of butadiene produced. DETAILED DESCRIPTION OF THE INVENTION

[0031] As will be shown in the Examples below, the present inventors have demonstrated that an unsaturated hydrocarbon compound having carbon-carbon double bonds at both ends, such as butadiene, can be produced by a three-step reaction using three types of enzymes as described below.

[0032] [ka]

[0033] In particular, the present inventors have demonstrated that phenylalanine ammonia-lyase (PAL) can be used as an enzyme involved in the production of pentadienoic acid from L-allylglycine.

[0034] Therefore, the present invention relates to a method for producing a chain unsaturated compound having at least two carbon-carbon double bonds, which comprises the following reaction steps:

[0035] [ka]

[0036] <Method for producing the third chain unsaturated carboxylic acid compound> As described above, the present invention provides a method for producing an open-chain unsaturated carboxylic acid compound represented by the formula (3) or a geometric isomer thereof (a third open-chain unsaturated carboxylic acid compound), comprising the step of eliminating a first amino group from an open-chain unsaturated carboxylic acid compound represented by the formula (2) or a geometric isomer thereof (a second open-chain unsaturated carboxylic acid compound), which has a first amino group and a first carbon-carbon double bond at a terminal, in the presence of phenylalanine ammonia-lyase, to form a second carbon-carbon double bond.

[0037] In the present invention, the "third chain unsaturated carboxylic acid compound" produced in the reaction means a chain unsaturated carboxylic acid compound having at least two carbon-carbon double bonds including a terminal carbon-carbon double bond and represented by the formula (3) or a geometric isomer thereof.

[0038] In the present invention, (A) in each chemical formula represents an optionally substituted linear hydrocarbon group having 0 to 5 carbon atoms. The term "linear hydrocarbon group having 0 carbon atoms" means that, in the compounds represented by each chemical formula and their geometric isomers, carbon atoms that are bonded via (A) are directly bonded without (A). Furthermore, when the linear hydrocarbon group having 2 to 5 carbon atoms is optionally substituted, at least one double bond may be formed between adjacent carbon atoms. Examples of the substituent that the hydrocarbon group in (A) may have include a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, a hydroxyl group, a halogen atom (e.g., fluorine, chlorine, bromine, iodine), a nitro group, a cyano group, an amino group, a carboxyl group, and a formyl group. Preferably, (A) in each chemical formula is a linear hydrocarbon group having 0 carbon atoms.

[0039] In the present invention, R in each chemical formula 1 and R 2 are each independently 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. Examples of "linear or branched alkyl groups having 1 to 5 carbon atoms" include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, and i-pentyl. Examples of "linear or branched alkoxy groups having 1 to 5 carbon atoms" include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, n-pentyloxy, i-pentyloxy, n-pentyloxy, and 1,2-dimethylpropoxy. R in each chemical formula 1 R is preferably a hydrogen atom. 2 is preferably a hydrogen atom or a methyl group.

[0040] (A) and R in each chemical formula 1 and R 2The combinations of are preferably a linear hydrocarbon group having 0 carbon atoms, a hydrogen group and a hydrogen group, or a linear hydrocarbon group having 0 carbon atoms, a hydrogen group and a methyl group. In the present invention, the "third chain unsaturated carboxylic acid compound" is preferably pentadienoic acid, 4-methylpentadienoic acid, or 3-methylpentadienoic acid.

[0041] The conditions for deaminating a second chain unsaturated carboxylic acid compound in the presence of the phenylalanine ammonia-lyase of the present invention may be any conditions that promote the deamination and produce a third chain unsaturated carboxylic acid compound, and a person skilled in the art can appropriately adjust and set the composition of the reaction solution, the pH of the reaction solution, the reaction temperature, the reaction time, etc.

[0042] For example, the reaction solution to which the phenylalanine ammonia-lyase of the present invention and its substrate, a second-chain unsaturated carboxylic acid compound, are added is not particularly limited as long as it does not interfere with the reaction, but is preferably a buffer solution of pH 6 to 8, more preferably a buffer solution of pH 6 to 7 containing potassium chloride and sodium phosphate.

[0043] The reaction temperature is not particularly limited as long as it does not interfere with the reaction, but is usually 20 to 40° C., and preferably 25 to 37° C. The reaction time is not particularly limited as long as it is a time that allows the unsaturated hydrocarbon compound to be produced, but is usually 30 minutes to 7 days, and preferably 12 hours to 2 days.

[0044] The third chain unsaturated carboxylic acid compound thus produced can be collected by appropriately utilizing known recovery and purification methods (distillation, chromatography, etc.). These methods may be carried out alone or in multiple stages in appropriate combination.

[0045] (phenylalanine ammonia-lyase) "Phenylalanine ammonia-lyase" is an enzyme registered under EC number 4.3.1.24 that catalyzes the reaction of phenylalanine as a substrate to produce cinnamic acid and ammonia. It is also known as PAL, tyrosine ammonia-lyase, phenylalanine deaminase, tyrosine ammonia-lyase, L-tyrosine ammonia-lyase, phenylalanine ammonium-lyase, or L-phenylalanine ammonia-lyase.

[0046] As will be shown in the Examples below, the phenylalanine ammonia lyase can be involved in the production of the above-mentioned third-chain unsaturated carboxylic acid compound, regardless of its origin. Therefore, the phenylalanine ammonia lyase according to the present invention is not particularly limited, and those derived from various organisms can be used. For example, phenylalanine ammonia lyase can be derived from Anabaena variabilis, Arabidopsis thaliana, Plagiochasma appendiculatum, Rhodotorula glutinis, Planctomyces brasiliensis, Oryza sativa, Bambusa oldhamii, Taxus chinensis, Nicotiana tabacum, Streptomyces maritimus, Salvia miltiorrhiza, and Solanum lycopersicum. Among these, phenylalanine ammonia lyase derived from Anabaena variabilis (phenylalanine ammonia lyase consisting of the amino acid sequence set forth in SEQ ID NO: 2) is preferred from the viewpoint of higher catalytic activity for producing a third-chain unsaturated carboxylic acid compound, as will be shown in the Examples below.

[0047] Furthermore, the identity of the phenylalanine ammonia-lyase of the present invention to the amino acid sequence set forth in SEQ ID NO:2 is preferably 15% or more (e.g., 16% or more, 17% or more, 18% or more, 19% or more), more preferably 20% or more (e.g., 30% or more, 40% or more), even more preferably 50% or more (e.g., 60% or more, 70% or more), more preferably 80% or more (e.g., 85% or more, 86% or more, 87% or more, 88% or more, 89% or more), and even more preferably 90% or more (e.g., 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more). The term "identity" to the amino acid sequence set forth in SEQ ID NO:2 refers to the percentage (%) of amino acids that match between the phenylalanine ammonia-lyase of the present invention and the amino acid sequence set forth in SEQ ID NO:2 relative to the total number of amino acids in the phenylalanine ammonia-lyase of the present invention.

[0048] Furthermore, the phenylalanine ammonia-lyase according to the present invention may be one in which a natural or non-natural (artificial) mutation has been introduced into the amino acid sequence set forth in SEQ ID NO: 2. That is, the phenylalanine ammonia-lyase according to the present invention also includes a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of phenylalanine ammonia-lyase (such as the amino acid sequence set forth in SEQ ID NO: 2). Here, the term "multiple" is not particularly limited, but typically refers to 2 to 200, preferably 2 to 150, more preferably 2 to 100, even more preferably 2 to 70, more preferably 2 to 50, even more preferably 2 to 30, more preferably 2 to 20, and even more preferably 2 to 10 (for example, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2).

[0049] Furthermore, as will be shown in the Examples below, the phenylalanine ammonia-lyase according to the present invention is preferably a phenylalanine ammonia-lyase having at least one of the following characteristics (1) to (5): (1) The amino acid at or corresponding to position 108 of the amino acid sequence set forth in SEQ ID NO: 2 is methionine, phenylalanine, or valine. (2) The amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is tryptophan. (3) The amino acid at position 219 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is isoleucine. (4) The amino acid at position 223 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is isoleucine. (5) The amino acid at position 104 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is alanine.

[0050] In the present invention, with respect to phenylalanine ammonia-lyase, the term "corresponding site" refers to a site that is aligned with the leucine at position 108, the phenylalanine at position 107, the leucine at position 104, the leucine at position 219, and the asparagine at position 223 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence of SEQ ID NO: 2 is aligned with the amino acid sequence of a phenylalanine ammonia-lyase derived from another species using nucleotide and amino acid sequence analysis software (GENETYX-MAC, Sequencher, etc.) or BLAST (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0051] Furthermore, with regard to the characteristics (1) to (5) above, the phenylalanine ammonia-lyase preferably has at least one of the following characteristics: the amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or a corresponding site is methionine; the amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or a corresponding site is tryptophan; and the amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or a corresponding site is phenylalanine; and more preferably the phenylalanine ammonia-lyase has at least one of the following characteristics: the amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or a corresponding site is methionine and the amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or a corresponding site is tryptophan.

[0052] Furthermore, such a phenylalanine ammonia-lyase having specific amino acids at each site may be a wild-type phenylalanine ammonia-lyase, or may be a mutant phenylalanine ammonia-lyase into which amino acid substitutions have been introduced so that the mutant has at least one of the characteristics (1) to (5) above.

[0053] More specifically, such mutants include any of the following (a) to (c): (a) A phenylalanine ammonia-lyase mutant comprising the amino acid sequence of SEQ ID NO: 2, into which at least one of the following amino acid substitutions (1) to (5) has been introduced: (1) The amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with methionine, phenylalanine, or valine; (2) the amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with tryptophan; (3) The amino acid at position 219 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with isoleucine; (4) The amino acid at position 223 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with isoleucine; (5) The amino acid at position 104 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with alanine. (b) A phenylalanine ammonia-lyase mutant comprising an amino acid sequence in which at least one of the amino acid substitutions (1) to (5) has been introduced into the amino acid sequence set forth in SEQ ID NO: 2, and further in which one or more amino acids have been substituted, deleted, added, and / or inserted at one or several sites other than the aforementioned sites. (c) A phenylalanine ammonia-lyase mutant comprising an amino acid sequence having at least 15% identity with the amino acid sequence set forth in SEQ ID NO: 2, and having at least one amino acid substitution selected from the amino acid sequences (1) to (5) introduced therein.

[0054] The "multiple" substituted, deleted, added, and / or inserted amino acids and the "identity" with the amino acid sequence set forth in SEQ ID NO: 2, including their preferred embodiments (scopes), are as described above. Furthermore, the phenylalanine ammonia-lyase mutant may be naturally occurring or non-naturally occurring (artificially occurring). That is, it also includes modified phenylalanine ammonia-lyases into which amino acid substitutions or the like have been artificially introduced.

[0055] Whether or not a phenylalanine ammonia-lyase or a natural or non-natural variant thereof has catalytic activity for producing a third-chain unsaturated carboxylic acid compound can be determined by a person skilled in the art by directly measuring the amount of the unsaturated carboxylic acid compound produced using a known method (e.g., chromatography-mass spectrometry).

[0056] Furthermore, other compounds may be directly or indirectly attached to the phenylalanine ammonia-lyase of the present invention. Such attachment is not particularly limited and may be at the genetic level or chemically attached. The site of attachment is also not particularly limited and may be either the amino terminus or the carboxyl terminus of the phenylalanine ammonia-lyase of the present invention, or both. Addition at the genetic level is achieved by using DNA encoding the phenylalanine ammonia-lyase of the present invention, with the reading frame of DNA encoding the other protein being added. The "other protein" thus added is not particularly limited. For the purpose of facilitating the purification of the phenylalanine ammonia-lyase of the present invention, preferred are purification tag proteins such as polyhistidine (His) tag protein, FLAG-tag protein (registered trademark, Sigma-Aldrich), and glutathione S-transferase (GST). For the purpose of facilitating the detection of the phenylalanine ammonia-lyase of the present invention, preferred are detection tag proteins such as fluorescent proteins such as GFP and chemiluminescent proteins such as luciferase. The chemical attachment may be a covalent bond or a non-covalent bond. The "covalent bond" is not particularly limited, and examples thereof include an amide bond between an amino group and a carboxyl group, an alkylamine bond between an amino group and an alkyl halide group, a disulfide bond between thiols, and a thioether bond between a thiol group and a maleimide group or an alkyl halide group. An example of a "non-covalent bond" is a biotin-avidin bond. Furthermore, for the purpose of facilitating detection of the phenylalanine ammonia-lyase of the present invention, suitable examples of the "other compound" chemically attached in this manner include fluorescent dyes such as Cy3 and rhodamine.

[0057] The phenylalanine ammonia-lyase of the present invention may be used in combination with other ingredients, which are not particularly limited and may include, for example, sterilized water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, protease inhibitor, and preservative.

[0058] <Method for producing a third chain unsaturated carboxylic acid compound via production of a second chain unsaturated carboxylic acid compound> As will be described in the Examples below, the present inventors have found that the second chain unsaturated carboxylic acid compound, which is a raw material for producing the above-mentioned third chain unsaturated carboxylic acid compound, can also be produced as shown in the following reaction.

[0059] [ka]

[0060] That is, the present inventors have found that the enzyme BesC can be used to oxidize the terminal propylamino group of an acyclic unsaturated carboxylic acid compound represented by formula (1) or a geometric isomer thereof (first acyclic unsaturated carboxylic acid compound), thereby cleaving the carbon-carbon bond in the group and forming a terminal carbon-carbon double bond, thereby producing a second acyclic unsaturated carboxylic acid compound represented by formula (2). Therefore, the present invention can also be embodied as a method for producing a third acyclic unsaturated carboxylic acid compound via the production of the second acyclic unsaturated carboxylic acid compound.

[0061] In the present invention, the "second chain unsaturated carboxylic acid compound" refers to a chain unsaturated carboxylic acid compound having a first amino group and a first terminal carbon-carbon double bond and represented by the formula (2) or a geometric isomer thereof. The "first chain unsaturated carboxylic acid compound" used as a raw material for producing the second chain unsaturated carboxylic acid compound refers to a chain unsaturated carboxylic acid compound having a first amino group and a terminal second amino group and represented by the formula (1) or a geometric isomer thereof.

[0062] In these compounds, (A) and R 1 The "second chain unsaturated carboxylic acid compound" is preferably L-allylglycine, L-(2-methylallyl)glycine, or L-(3-methylallyl)glycine. The "first chain unsaturated carboxylic acid compound" is preferably L-lysine, 4-methyllysine, or 3-methyllysine.

[0063] The conditions for forming a carbon-carbon double bond at the end of a first chain-like unsaturated carboxylic acid compound in the presence of BesC according to the present invention may be any conditions that promote the formation of the double bond and produce a second chain-like unsaturated carboxylic acid compound, and a person skilled in the art would be able to appropriately adjust and set the composition of the reaction solution, the pH of the reaction solution, the reaction temperature, the reaction time, etc.

[0064] For example, the reaction solution to which BesC according to the present invention and its substrate, the first chain unsaturated carboxylic acid compound, are added is not particularly limited as long as it does not interfere with the reaction, but is preferably a buffer solution of pH 6 to 8, more preferably a buffer solution containing potassium chloride and sodium phosphate of pH 6 to 7. Furthermore, from the viewpoint of further facilitating the reaction, it is preferable that the solution contains iron sulfate.

[0065] The reaction temperature is not particularly limited as long as it does not interfere with the reaction, but is usually 20 to 40° C., and preferably 25 to 37° C. Furthermore, the reaction time is not particularly limited as long as it is a time that allows the unsaturated hydrocarbon compound to be produced, and is usually 30 minutes to 7 days, and preferably 12 hours to 2 days.

[0066] The second chain unsaturated carboxylic acid compound thus produced can be collected by appropriately utilizing known recovery and purification methods (distillation, chromatography, etc.). These methods may be carried out alone or in multiple stages in appropriate combination.

[0067] The first chain unsaturated carboxylic acid compound used as the raw material can be purchased as a commercially available product, as shown in the Examples below. Alternatively, a person skilled in the art can synthesize the compound by appropriately referring to known synthesis methods (for example, the method described in the fermentation method for producing L-lysine (JPH0530985A)).

[0068] (BesC) "BesC" is an enzyme involved in β-ethynylserine biosynthesis (Bes). This enzyme is a terminal alkene-synthesizing enzyme that catalyzes the reaction of oxidizing the terminal propylamino group, thereby cleaving the carbon-carbon bond in the group and forming a terminal carbon-carbon double bond (Non-Patent Document 2).

[0069] The origin of BesC is not particularly limited, and various organisms can be used as long as it has catalytic activity that promotes the production of a second-chain unsaturated carboxylic acid compound. Examples of BesC sources include Pseudomonas fluorescens and Streptomyces cattleya. Among these, BesC derived from Pseudomonas fluorescens (BesC consisting of the amino acid sequence set forth in SEQ ID NO: 11) is preferred.

[0070] Furthermore, the identity of the BesC of the present invention to the amino acid sequence set forth in SEQ ID NO: 11 is preferably 15% or more (e.g., 16% or more, 17% or more, 18% or more, 19% or more), more preferably 20% or more (e.g., 30% or more, 40% or more), even more preferably 50% or more (e.g., 60% or more, 70% or more), more preferably 80% or more (e.g., 85% or more, 86% or more, 87% or more, 88% or more, 89% or more), and even more preferably 90% or more (e.g., 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more). The term "identity" to the amino acid sequence set forth in SEQ ID NO: 11 refers to the percentage (%) of amino acids identical between the BesC of the present invention and the amino acid sequence set forth in SEQ ID NO: 11 relative to the total number of amino acids in the BesC of the present invention.

[0071] Furthermore, BesC according to the present invention may be one in which a natural or non-natural (artificial) mutation has been introduced into the amino acid sequence of SEQ ID NO: 11. That is, BesC according to the present invention also includes proteins consisting of the amino acid sequence of BesC (such as the amino acid sequence of SEQ ID NO: 11) in which one or more amino acids have been substituted, deleted, added, and / or inserted. Here, the term "multiple" is not particularly limited, but typically refers to 2 to 80, preferably 2 to 70, more preferably 2 to 60, even more preferably 2 to 50, more preferably 2 to 40, even more preferably 2 to 30, more preferably 2 to 20, and even more preferably 2 to 10 (e.g., 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2).

[0072] Whether BesC or a natural or non-natural variant thereof has catalytic activity to produce a second chain unsaturated carboxylic acid compound can be determined by a person skilled in the art by directly measuring the amount of the unsaturated carboxylic acid compound produced using a known method (e.g., chromatography-mass spectrometry).

[0073] Furthermore, similar to the phenylalanine ammonia-lyase of the present invention, other compounds may be added directly or indirectly to the BesC of the present invention, and further, similar to the phenylalanine ammonia-lyase of the present invention, the BesC of the present invention may be used in combination with other components.

[0074] <Method of producing chain unsaturated hydrocarbon compound> As will be shown in the Examples below, the present inventors have also found that a chain unsaturated hydrocarbon compound having carbon-carbon double bonds at both ends can be produced from the third chain unsaturated carboxylic acid compound described above.

[0075] That is, as described above, the present invention may take the form of a production method shown below, which includes the steps of: producing a third chain unsaturated carboxylic acid compound represented by the following formula (3) from a second chain unsaturated carboxylic acid compound represented by the following formula (2) by using phenylalanine ammonia lyase; and producing a chain unsaturated hydrocarbon compound represented by the following formula (4) or a geometric isomer thereof (hereinafter simply referred to as "chain unsaturated hydrocarbon compound") from the unsaturated carboxylic acid compound by using ferulic acid decarboxylase to eliminate the carboxyl group.

[0076] [ka]

[0077] [ka]

[0078] In the present invention, the "chain unsaturated hydrocarbon compound" produced in the reaction means a chain unsaturated hydrocarbon compound having carbon-carbon double bonds at both ends and represented by the formula (4) or a geometric isomer thereof.

[0079] In the compound, (A) and R 1The above-mentioned compounds, including their preferred embodiments, are as described above, and the "chain unsaturated hydrocarbon compound" is preferably butadiene or isoprene.

[0080] The conditions for decarboxylating a third chain unsaturated carboxylic acid compound in the presence of the ferulic acid decarboxylase of the present invention may be any conditions that promote the decarboxylation and produce an unsaturated hydrocarbon compound, and a person skilled in the art can appropriately adjust and set the composition of the reaction solution, the pH of the reaction solution, the reaction temperature, the reaction time, etc.

[0081] For example, the reaction solution to which the ferulic acid decarboxylase of the present invention and its substrate, an unsaturated hydrocarbon dicarboxylic acid compound, are added is not particularly limited as long as it does not interfere with the reaction, but is preferably a buffer solution of pH 6 to 8, more preferably a buffer solution of pH 6 to 7 containing potassium chloride and sodium phosphate. Furthermore, from the viewpoint of facilitating the reaction, prenylated flavin mononucleotide (prFMN) or its isomer (prFMN) may be used. ketimine , prFMN iminiu It is preferable that prFMN and its isomers are contained therein (see Non-Patent Document 1 for details).

[0082] The reaction temperature is not particularly limited as long as it does not interfere with the reaction, but is usually 20 to 40° C., and preferably 25 to 37° C. The reaction time is not particularly limited as long as it is a time that allows the unsaturated hydrocarbon compound to be produced, but is usually 30 minutes to 7 days, and preferably 12 hours to 2 days.

[0083] Furthermore, since the chain-like unsaturated hydrocarbon compounds produced under such conditions are generally prone to vaporization, they can be collected by known methods for recovering and purifying volatile gases. Examples of such collection methods include gas stripping, fractional distillation, adsorption, desorption, pervaporation, desorption of unsaturated hydrocarbon compounds adsorbed on a solid phase from the solid phase by heat or vacuum, extraction with a solvent, or chromatography (e.g., gas chromatography). Furthermore, even when the unsaturated hydrocarbon compounds produced are liquid, they can be collected by appropriately utilizing known recovery and purification methods (distillation, chromatography, etc.). Furthermore, these methods may be carried out alone or in appropriate combinations in multiple stages.

[0084] (ferulic acid decarboxylase) "Ferulic acid decarboxylase" is an enzyme registered under EC number 4.1.1.102, and generally refers to an enzyme that catalyzes the reaction of decarboxylating ferulic acid to produce 4-vinylguaiacol (4VG).

[0085] In the present invention, the "ferulic acid decarboxylase" is not particularly limited as long as it has the activity of catalyzing a reaction that cleaves a carboxyl group from a third-chain unsaturated carboxylic acid compound to produce a chain unsaturated hydrocarbon compound, and various biologically derived ferulic acid decarboxylases can be used. Examples include proteins that correspond to "ferulic acid decarboxylases" on UNIPROT, such as those shown in Tables 1 to 6 of Patent Document 3. Furthermore, preferred examples of the ferulic acid decarboxylases of the present invention include ferulic acid decarboxylases derived from Saccharomyces and ferulic acid decarboxylases derived from Aspergillus oryzae (UNIPROT ID: A2QHE5, etc.), and more preferred examples include ferulic acid decarboxylases derived from Saccharomyces. The term "Saccharomyces" refers to bacteria belonging to the genus Saccharomyces, such as Saccharomyces cerevisiae, Saccharomyces kudriavzevii, Saccharomyces eubayanus, Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces florentinus, Saccharomyces kluyveri, Saccharomyces martiniae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, and Saccharomyces zonatus.

[0086] Examples of ferulic acid decarboxylases derived from Saccharomyces include FDC (ferulic acid decarboxylase consisting of the amino acid sequence set forth in UNIPROT ID: Q03034, SEQ ID NO: 13) derived from Saccharomyces cerevisiae (ATCC 204508 / S288c strain) (baker's yeast), as well as proteins derived from Saccharomyces that correspond to "Ferulic acid decarboxylase" on UNIPROT, and more specifically, the ferulic acid decarboxylases listed in Table 1 below. It should be understood that changes in the amino acid sequence of proteins can occur in nature due to mutations in nucleotide sequences.

[0087] [Table 1]

[0088] Furthermore, the identity of the ferulic acid decarboxylase of the present invention with the amino acid sequence set forth in SEQ ID NO: 13 is preferably 15% or more (e.g., 16% or more, 17% or more, 18% or more, 19% or more), more preferably 20% or more (e.g., 30% or more, 40% or more), even more preferably 50% or more (e.g., 60% or more, 70% or more), more preferably 80% or more (e.g., 85% or more, 86% or more, 87% or more, 88% or more, 89% or more), and even more preferably 90% or more (e.g., 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more). The term "identity" with the amino acid sequence set forth in SEQ ID NO: 13 refers to the percentage (%) of amino acids that match between the ferulic acid decarboxylase of the present invention and the amino acid sequence set forth in SEQ ID NO: 13 relative to the total number of amino acids in the ferulic acid decarboxylase of the present invention.

[0089] The ferulic acid decarboxylase of the present invention may also be one in which a mutation has been introduced into the amino acid sequence of SEQ ID NO: 13. That is, the ferulic acid decarboxylase of the present invention also includes proteins consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of ferulic acid decarboxylase (such as the amino acid sequence of SEQ ID NO: 13). Here, the term "multiple" is not particularly limited, but typically refers to 2 to 150, preferably 2 to 100, more preferably 2 to 80, even more preferably 2 to 50, even more preferably 2 to 30, even more preferably 2 to 20, and even more preferably 2 to 10 (e.g., 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2). Such ferulic acid decarboxylase mutants may be naturally occurring or non-naturally occurring (artificially occurring). That is, modified ferulic acid decarboxylases in which amino acid substitutions or the like have been artificially introduced are also included.

[0090] Whether or not ferulic acid decarboxylase or a natural or non-natural variant thereof has catalytic activity to produce linear unsaturated hydrocarbon compounds can be determined by a person skilled in the art, for example, by directly measuring the amount of unsaturated hydrocarbon compounds by gas chromatography-mass spectrometry (GC-MS), as shown in the Examples below.

[0091] Similarly to the phenylalanine ammonia-lyase of the present invention, the ferulic acid decarboxylase of the present invention may have other compounds added thereto directly or indirectly.Furthermore, similar to the phenylalanine ammonia-lyase of the present invention, the ferulic acid decarboxylase of the present invention may be used in combination with other components.

[0092] <DNA encoding the enzyme of the present invention and vector containing said DNA> Next, DNAs encoding the enzymes of the present invention (phenylalanine ammonia-lyase of the present invention, BesC of the present invention, and ferulic acid decarboxylase of the present invention) will be described. By introducing such DNAs, host cells can be transformed to produce various enzymes of the present invention in the cells, and ultimately, it becomes possible to produce a third chain unsaturated carboxylic acid compound or a chain unsaturated hydrocarbon compound.

[0093] The DNA of the present invention may be natural DNA, DNA into which mutations have been artificially introduced, or DNA consisting of an artificially designed nucleotide sequence, as long as it encodes the enzyme of the present invention. Furthermore, there are no particular limitations on its form, and it includes cDNA, genomic DNA, and chemically synthesized DNA. These DNAs can be prepared using routine methods known to those skilled in the art. Genomic DNA can be prepared, for example, by extracting genomic DNA from various bacteria, constructing a genomic library (vectors that can be used include plasmids, phages, cosmids, BACs, and PACs), expanding the library, and performing colony hybridization or plaque hybridization using a probe prepared based on the nucleotide sequence of the gene encoding the enzyme of the present invention (e.g., the nucleotide sequence set forth in SEQ ID NO: 1). Alternatively, the DNA can be prepared by PCR using primers specific to the gene encoding the enzyme of the present invention. Alternatively, cDNA can be prepared by synthesizing cDNA based on mRNA extracted from various bacteria, inserting it into a vector such as λZAP to create a cDNA library, expanding it, and performing colony hybridization or plaque hybridization as described above, or by performing PCR.

[0094] Those skilled in the art can then introduce mutations encoding the above-mentioned amino acid substitutions into the DNA prepared in this manner, if necessary, using known site-specific mutagenesis methods, such as the Kunkel method (Kunkel, TA, Proc Natl Acad Sci USA, 1985, Vol. 82, No. 2, pp. 488-492) and the splicing-by-overlap-extension (SOE) PCR method (Ho, SN, Hunt, HD, Horton, RM, Pullen, JK, and Pease, LR, Gene, 1989, Vol. 77, pp. 51-59).

[0095] Furthermore, a person skilled in the art can artificially design a nucleotide sequence encoding a protein into which the above-mentioned amino acid substitutions have been introduced, and chemically synthesize the DNA of the present invention using an automated nucleic acid synthesizer based on the sequence information.

[0096] Furthermore, from the viewpoint of further improving the expression efficiency of the encoded decarboxylase of the present invention in host cells, the DNA of the present invention may also take the form of DNA encoding the enzyme of the present invention, in which codons have been optimized to suit the type of host cell.

[0097] Furthermore, the present invention may take the form of a vector into which the aforementioned DNA has been inserted so that the DNA can be replicated in a host cell.

[0098] In the present invention, a "vector" refers to a self-replicating vector, i.e., a vector that exists as an extrachromosomal independent entity and whose replication does not depend on chromosomal replication, and can be constructed, for example, based on a plasmid. Alternatively, a vector may be one that, when introduced into a host cell, is integrated into the genome of the host cell and replicated together with the chromosome into which it has been integrated.

[0099] Examples of such vectors include plasmids and phage DNA. Examples of plasmids include Escherichia coli-derived plasmids (pET22, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), and Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.). Examples of phage DNA include λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.). Furthermore, if the host cell is derived from an insect, an insect virus vector such as baculovirus can be used as a vector of the present invention. If the host cell is derived from a plant, a T-DNA vector can be used as a vector of the present invention. If the host cell is derived from an animal, an animal virus vector such as a retrovirus or adenovirus vector can be used as a vector. Furthermore, procedures and methods commonly used in the field of genetic engineering can be used for constructing the vector of the present invention. For example, to insert the DNA of the present invention into a vector, a method may be employed in which the purified DNA is first cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or multicloning site of an appropriate vector, and then ligated to the vector.

[0100] Furthermore, the vector of the present invention may be in the form of an expression vector containing the enzyme of the present invention encoded by the DNA in a state capable of being expressed in host cells. In order to express the enzyme of the present invention by introducing the "expression vector" of the present invention into host cells, it is desirable to include, in addition to the DNA, a DNA sequence that controls the expression and a genetic marker for selecting transformed host cells. DNA sequences that control expression include promoters, enhancers, splicing signals, poly(A) addition signals, ribosome binding sequences (SD sequences), and terminators. The promoter is not particularly limited as long as it exhibits transcriptional activity in host cells, and can be obtained as a DNA sequence that controls the expression of a gene encoding a protein of either the same or a different species as the host cell. Furthermore, in addition to the DNA sequence that controls expression, the vector may also contain a DNA sequence that induces expression. When the host cell is a bacterium, an example of such an expression-inducing DNA sequence is the lactose operon, which can induce the expression of a gene located downstream by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG). The genetic marker in the present invention may be appropriately selected depending on the method for selecting transformed host cells, and for example, a gene encoding drug resistance or a gene complementing auxotrophy can be used.

[0101] Each vector may contain one type of DNA encoding the enzyme of the present invention, or multiple types of DNA may be inserted into one vector. When multiple types of DNA are inserted into a single vector, it is preferable that these DNAs form an operon. Here, an "operon" is a nucleic acid sequence unit consisting of one or more genes transcribed under the control of the same promoter.

[0102] In the present invention, the embodiment of such a vector is: a vector into which DNA encoding the phenylalanine ammonia-lyase of the present invention has been inserted; a combination of a vector into which the DNA encoding BesC of the present invention has been inserted and a vector into which the DNA encoding the phenylalanine ammonia-lyase of the present invention has been inserted; A combination of a vector into which DNA encoding the phenylalanine ammonia-lyase of the present invention has been inserted and a vector into which DNA encoding the ferulic acid decarboxylase of the present invention has been inserted, or Examples include a combination of a vector into which DNA encoding BesC of the present invention has been inserted, a vector into which DNA encoding phenylalanine ammonia-lyase of the present invention has been inserted, and a vector into which DNA encoding ferulic acid decarboxylase of the present invention has been inserted. Furthermore, a vector into which a DNA encoding BesC according to the present invention and a DNA encoding a phenylalanine ammonia-lyase according to the present invention are inserted, A vector into which a DNA encoding the phenylalanine ammonia-lyase of the present invention and a ferulic acid decarboxylase of the present invention are inserted, or Also included is a vector into which a DNA encoding BesC according to the present invention, a DNA encoding a phenylalanine ammonia-lyase according to the present invention, and a ferulic acid decarboxylase according to the present invention have been inserted.

[0103] The DNA or vector of the present invention may be used in combination with other components, including, but not limited to, sterilized water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, DNase inhibitor, and preservative.

[0104] <Agent for Promoting the Production of Third Chain Unsaturated Carboxylic Acid Compound or Chain Unsaturated Hydrocarbon Compound> As described above, by using the enzyme of the present invention, the DNA encoding the enzyme, or the vector into which the DNA has been inserted, it is possible to promote the production of a third chain unsaturated carboxylic acid compound or a chain unsaturated hydrocarbon compound.

[0105] Therefore, the present invention provides an agent for promoting the production of a third chain unsaturated carboxylic acid compound or a chain unsaturated hydrocarbon compound, which comprises an enzyme of the present invention, DNA encoding the enzyme, or a vector into which the DNA has been inserted.

[0106] More specifically, the following aspects can be mentioned: an agent for promoting the production of a third-chain unsaturated carboxylic acid compound from a second-chain unsaturated carboxylic acid compound, the agent comprising the phenylalanine ammonia-lyase of the present invention, a DNA encoding the enzyme, or a vector into which the DNA has been inserted; An agent for promoting the production of a third chain unsaturated carboxylic acid compound from a first chain unsaturated carboxylic acid compound, comprising BesC according to the present invention, DNA encoding the enzyme or a vector into which the DNA has been inserted, and phenylalanine ammonia-lyase according to the present invention, DNA encoding the enzyme or a vector into which the DNA has been inserted; an agent for promoting the production of a third chain unsaturated carboxylic acid compound from a first chain unsaturated carboxylic acid compound, the agent comprising a vector into which a DNA encoding the BesC of the present invention and a DNA encoding the phenylalanine ammonia-lyase of the present invention are inserted; an agent for promoting the production of a chain unsaturated hydrocarbon compound from a second chain unsaturated carboxylic acid compound, the agent comprising the phenylalanine ammonia-lyase of the present invention, a DNA encoding the enzyme, or a vector into which the DNA has been inserted, and the ferulic acid decarboxylase of the present invention, a DNA encoding the enzyme, or a vector into which the DNA has been inserted; an agent for promoting the production of a chain unsaturated hydrocarbon compound from a second chain unsaturated carboxylic acid compound, the agent comprising a vector into which a DNA encoding the phenylalanine ammonia-lyase of the present invention and a DNA encoding the ferulic acid decarboxylase of the present invention are inserted; an agent for promoting the production of a chain unsaturated hydrocarbon compound from a first chain unsaturated carboxylic acid compound, the agent comprising: BesC according to the present invention, a DNA encoding the enzyme or a vector into which the DNA has been inserted; phenylalanine ammonia-lyase according to the present invention, a DNA encoding the enzyme or a vector into which the DNA has been inserted; and ferulic acid decarboxylase according to the present invention, a DNA encoding the enzyme or a vector into which the DNA has been inserted; An agent for promoting the production of a chain-like unsaturated hydrocarbon compound from a first chain-like unsaturated carboxylic acid compound, comprising a vector into which DNA encoding BesC of the present invention, DNA encoding phenylalanine ammonia-lyase of the present invention, and DNA encoding ferulic acid decarboxylase of the present invention are inserted.

[0107] Such an agent may be any agent containing the enzyme of the present invention, or may be used in combination with other components, such as, but not limited to, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, protease inhibitor, DNase inhibitor, and preservative.

[0108] The present invention can also provide a kit containing such an agent. In the kit of the present invention, the agent may be contained in the form of a host cell, described below, transformed with the DNA of the present invention. In addition to such an agent, the kit of the present invention may also contain various substrates (the first chain unsaturated carboxylic acid compound, the second chain unsaturated carboxylic acid compound), host cells for introducing the DNA of the present invention, a medium for culturing the host cells, and instructions for using them. Such instructions are instructions for using the agent of the present invention in the method for producing the third chain unsaturated carboxylic acid compound or the chain unsaturated hydrocarbon compound. The instructions may include, for example, experimental techniques and conditions for the production method of the present invention, and information about the agent of the present invention (for example, information such as a vector map showing the nucleotide sequence of the vector, sequence information of the enzyme of the present invention, information on the origin and properties of the host cell, and culture conditions of the host cell).

[0109] <Host cells into which DNA encoding the enzyme of the present invention has been introduced> Next, a host cell into which the DNA or vector of the present invention has been introduced will be described. Using a host cell transformed by introducing the above-mentioned DNA or vector, it becomes possible to produce the enzyme of the present invention, and further, it becomes possible to produce a third chain unsaturated carboxylic acid compound or a chain unsaturated hydrocarbon compound.

[0110] The host cells into which the DNA or vector of the present invention is introduced are not particularly limited, and examples thereof include microorganisms (Escherichia coli, budding yeast, fission yeast, Bacillus subtilis, actinomycetes, filamentous fungi, etc.), plant cells, insect cells, and animal cells. However, from the viewpoint of exhibiting high proliferation in a relatively inexpensive medium in a short period of time and thus contributing to the highly productive production of a third chain unsaturated carboxylic acid compound or a chain unsaturated hydrocarbon compound, it is preferable to use a microorganism as the host cell, and it is more preferable to use Escherichia coli.

[0111] Furthermore, the host cells into which the DNA or vector of the present invention is introduced are preferably cells that harbor flavin prenyltransferase, from the viewpoint of inducing prenylation of flavin mononucleotide (FMN) and producing prFMN or its isomer, which contributes to improving the productivity of linear unsaturated hydrocarbon compounds.

[0112] Furthermore, the host cells into which the DNA or vector of the present invention is introduced are preferably Escherichia coli C41(DE3) cells, from the viewpoints that they are easy to handle and capable of expressing toxic proteins.

[0113] The DNA or vector of the present invention can be introduced according to methods commonly used in this field. For example, methods for introduction into microorganisms such as Escherichia coli include the heat shock method, electroporation, spheroplast method, and lithium acetate method, methods for introduction into plant cells include methods using Agrobacterium and particle gun method, methods for introduction into insect cells include methods using baculovirus and electroporation method, and methods for introduction into animal cells include the calcium phosphate method, lipofection, and electroporation method.

[0114] The DNA etc. introduced into the host cell in this way may be maintained within the host cell by being randomly inserted into the genomic DNA, or by homologous recombination, or if it is a vector, it may be replicated and maintained as an independent entity outside the genomic DNA.

[0115] <Method for producing a third chain unsaturated carboxylic acid compound or a chain unsaturated hydrocarbon compound using a host cell> As described above, a third chain unsaturated carboxylic acid compound or a chain unsaturated hydrocarbon compound can be produced by culturing a host cell transformed to express the enzyme of the present invention. Thus, the present invention also provides a method for producing a third chain unsaturated carboxylic acid compound or a chain unsaturated hydrocarbon compound, which comprises the steps of culturing a host cell into which a DNA or vector encoding the enzyme of the present invention has been introduced, and collecting the third chain unsaturated carboxylic acid compound or the chain unsaturated hydrocarbon compound produced in the host cell and / or the culture thereof.

[0116] The "host cell transformed to express the enzyme of the present invention" is as described above. Furthermore, the combination of the enzyme of the present invention, the DNA encoding the enzyme, and the vector into which the DNA has been inserted, which are to be expressed or retained in such a host cell, can be appropriately designed and modified by a person skilled in the art, taking into account the combination of the starting material (substrate) and the final product, based on the examples shown above in <DNA encoding the enzyme of the present invention and the vector having the DNA> and <Agent for promoting the production of a third chain unsaturated carboxylic acid compound or a chain unsaturated hydrocarbon compound>.

[0117] The culture conditions for the cells are as described below, but it is preferable that various substrates (a first chain unsaturated carboxylic acid compound, a second chain unsaturated carboxylic acid compound) are added to the medium. The culture temperature can be appropriately designed and changed depending on the type of host cell used, but is usually 20 to 40°C, preferably 25 to 37°C.

[0118] In the present invention, the term "culture" refers to a medium containing grown host cells, secretory products of the host cells, metabolic products of the host cells, etc., obtained by culturing host cells in the medium, and includes dilutions and concentrates thereof. There are no particular limitations on the method for collecting the third chain unsaturated carboxylic acid compound or chain unsaturated hydrocarbon compound from such host cells and / or culture, and the collection can be carried out using the known recovery and purification methods described above. The collection time is adjusted appropriately depending on the type of host cells used, and may be any time that allows the third chain unsaturated carboxylic acid compound or chain unsaturated hydrocarbon compound to be produced, but is usually 30 minutes to 7 days, and preferably 12 hours to 2 days.

[0119] <Method for producing a phenylalanine ammonia-lyase variant according to the present invention> As will be shown in the Examples below, by culturing host cells into which DNA encoding the phenylalanine ammonia-lyase variant of the present invention has been introduced, the variant can be produced within the host cells.

[0120] Therefore, the present invention can also provide a method for producing a phenylalanine ammonia-lyase variant, the method comprising the steps of culturing a host cell into which DNA encoding the phenylalanine ammonia-lyase variant of the present invention or a vector containing the DNA has been introduced, and collecting the protein expressed in the host cell.

[0121] In the present invention, the conditions for "culturing host cells" may be any conditions that allow the host cells to produce the phenylalanine ammonia-lyase variant of the present invention. Those skilled in the art can appropriately adjust and set the temperature, whether or not air is added, the oxygen concentration, the carbon dioxide concentration, the pH of the medium, the culture temperature, the culture time, the humidity, and the like, depending on the type of host cells, the medium used, and the like.

[0122] Such a medium may contain any substance that can be utilized by the host cells, such as a carbon source, a nitrogen source, a sulfur source, inorganic salts, metals, peptone, yeast extract, meat extract, casein hydrolysate, serum, etc. Furthermore, such a medium may be supplemented with, for example, IPTG for inducing expression of the DNA encoding the phenylalanine ammonia-lyase variant of the present invention, an antibiotic (e.g., ampicillin) corresponding to a drug resistance gene that can be encoded by a vector of the present invention, or a nutrient (e.g., arginine, histidine) corresponding to a gene that complements an auxotrophy that can be encoded by a vector of the present invention.

[0123] Methods for "collecting the protein expressed in the host cells" from the host cells cultured in this manner include, for example, recovering the host cells from the medium by filtration, centrifugation, or the like, treating the recovered host cells by cell lysis, grinding, or pressure crushing, and further purifying and concentrating the protein expressed in the host cells by ultrafiltration, salting out, solvent precipitation such as ammonium sulfate precipitation, chromatography (e.g., gel chromatography, ion exchange chromatography, affinity chromatography), or the like. Furthermore, when the phenylalanine ammonia-lyase variant of the present invention has the aforementioned purification tag protein attached, it can also be purified and collected using a substrate to which the tag protein adsorbs. Furthermore, these purification and concentration methods may be performed alone or in appropriate combinations in multiple steps.

[0124] Furthermore, the phenylalanine ammonia-lyase variants of the present invention are not limited to the above biological synthesis, but can also be produced using the DNA or the like of the present invention and a cell-free protein synthesis system. There are no particular limitations on such cell-free protein synthesis systems, and examples include wheat germ-derived, Escherichia coli-derived, rabbit reticulocyte-derived, and insect cell-derived synthesis systems. Furthermore, those skilled in the art can also chemically synthesize the phenylalanine ammonia-lyase variants of the present invention using commercially available peptide synthesizers or the like.

[0125] The present invention also provides a method for producing a phenylalanine ammonia-lyase variant having enhanced catalytic activity for producing a third chain unsaturated carboxylic acid compound from a second chain unsaturated carboxylic acid compound, the method comprising the step of introducing at least one of the following amino acid substitutions (1) to (5) into phenylalanine ammonia-lyase: (1) replacing the amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position with methionine, phenylalanine, or valine; (2) replacing the amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position with tryptophan; (3) replacing the amino acid at position 219 of the amino acid sequence set forth in SEQ ID NO: 2 or a corresponding position with isoleucine; (4) Substitution of the amino acid at or corresponding to position 223 of the amino acid sequence set forth in SEQ ID NO: 2 with isoleucine; (5) The amino acid at position 104 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with alanine.

[0126] The term "variant phenylalanine ammonia-lyase having enhanced catalytic activity for producing a third chain unsaturated carboxylic acid compound from a second chain unsaturated carboxylic acid compound" refers to a phenylalanine ammonia-lyase in which the introduction of the amino acid substitution has enhanced catalytic activity for producing a third chain unsaturated carboxylic acid compound compared to before the introduction, and the comparison targets are usually the phenylalanine ammonia-lyases derived from the various organisms listed above and their natural variants.

[0127] "Introduction of amino acid substitutions" in phenylalanine ammonia-lyase can be achieved by modifying the encoding DNA. As described above, such "DNA modification" can be appropriately performed by methods known to those skilled in the art, such as site-directed mutagenesis or chemical synthesis of DNA based on modified sequence information. Furthermore, "introduction of amino acid substitutions" can also be achieved using chemical peptide synthesis methods, as described above.

[0128] Whether or not the catalytic activity for producing an olefin compound is enhanced by introducing such amino acid substitutions can be evaluated by chromatography-mass spectrometry, etc., as described above. Furthermore, the catalytic activity of the phenylalanine ammonia-lyase variant produced in this manner for producing a third chain unsaturated carboxylic acid compound is preferably 1.2 times or more (1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more), more preferably 2 times or more, even more preferably 3 times or more, more preferably 4 times or more, and even more preferably 5 times or more, compared to that of the phenylalanine ammonia-lyase consisting of the amino acid sequence set forth in SEQ ID NO: 2. [Example]

[0129] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0130] The present inventors have conceived the following reaction scheme in which butadiene is produced from L-lysine as a starting material in a three-step reaction using three enzymes (BesC, PAL, and FDC).

[0131] [ka]

[0132] Therefore, a demonstration experiment was carried out for this reaction scheme as follows.

[0133] Example 1 <Preparation of Plasmid Vector> First, to efficiently express Pseudomonas fluorescens-derived BesC, Arabidopsis thaliana-derived PAL, and Saccharomyces cerevisiae-derived FDC in E. coli, modified nucleotide sequences were designed taking into account codon usage in E. coli. DNA consisting of these modified nucleotide sequences was then chemically synthesized using standard methods. The resulting DNA was then ligated to the pET22b(+) vector (Novagen) using the Gibson Assembly method (using the NEBuilder HiFi DNA Assembly Master Mix® kit from New England Biolabs) to prepare plasmid vectors (BesC vector, PAL vector, and FDC vector 1) capable of expressing each of the wild-type genes in E. coli. Similarly, a plasmid vector (UbiX vector) capable of expressing wild-type UbiX in E. coli was prepared by linking DNA amplified by PCR from the gene (SEQ ID NO: 15) encoding flavin prenyltransferase (hereinafter also referred to as "UbiX") from an Escherichia coli (K-12) strain to the pColADuet vector (Novagen) using the Gibson Assembly method.

[0134] For the amino acid sequences of the enzymes used in Example 1 and Example 2 described below, and the DNA sequences encoding them (wild-type sequences and sequences modified taking into account the frequency of codon usage in E. coli), please refer to the sequences listed in the sequence numbers in Table 2 below.

[0135] [Table 2]

[0136] <Preparation of medium for enzyme expression and measurement of enzyme activity> The vectors prepared as described above (5 μg of BesC vector or 5 μg of PAL vector) were introduced into Escherichia coli C41(DE3) strain (Lucigen Corporation, 100 μL) by heat shock to prepare transformants expressing wild-type BesC or PAL. These transformants were then cultured for 6 hours in LB medium supplemented with ampicillin. Note that the growth of these transformants reaches a plateau after this 6-hour culture (preculture). Therefore, the bacterial mass at the start of the enzymatic reaction described below is uniform among these transformants.

[0137] In addition, 12 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, 20 g / L lactose, and 100 mg / L ampicillin were added to prepare a medium for enzyme expression.

[0138] 1 mL of the 6-hour cultured E. coli and 100 mL of the enzyme expression medium were added to a 500 mL baffled Erlenmeyer flask and cultured for an additional 18 hours at 37°C with a shaking speed of 180 rpm. After the culture, the medium was centrifuged at 5,000 rpm for 10 minutes, the supernatant was removed, and the protein extract solution B-PER was added. TM 10 mL of Bacterial Cell Lysis Reagent (Thermo Fisher Scientific) was added to suspend the pellet, and the mixture was shaken in an ice bath at a shaking speed of 60 rpm, followed by centrifugation at 15,000 rpm for 15 minutes to prepare various cell lysates.

[0139] Similarly, the prepared vectors (5 μg of FDC vector 1 and 5 μg of UbiX vector) were introduced into Escherichia coli C41(DE3) strain (Lucigen Corporation, 100 μL) by heat shock to prepare transformants co-expressing wild-type FDC and UbiX. The transformants were cultured for 6 hours in LB medium supplemented with ampicillin and kanamycin. The same enzyme reaction medium was prepared as above, supplemented with 50 mg / L kanamycin, and a cell lysate was prepared in the same manner.

[0140] Then, 100 μL of each cell lysate, 100 μL of 1 M phosphate buffer (pH 6.8), and L-lysine (to a final concentration of 10 mM) were added to 10 mL vials for headspace gas chromatography mass spectrometry (HS / GSMS). Ultrapure water was added to make a final volume of 1 mL. Immediately after that, the caps of the vials were closed, and the enzyme reaction was carried out at 37°C and a shaking speed of 180 rpm. 18 hours after the start of the reaction, the peak area representing the amount of 1,3-butadiene produced in the headspace of the vials was measured using a GC-MS (product name: GCMS-QP Ultra, manufactured by Shimadzu Corporation).

[0141] As a result, although not shown in the figure, butadiene was not detected when L-lysine was not added, but when L-lysine was added, the production of 1,3-butadiene was confirmed.

[0142] Example 2 As shown in Example 1, butadiene was confirmed to be produced from L-lysine by combining various wild-type enzymes. Therefore, in order to further improve this catalytic activity, PALs derived from other hosts were examined as PALs that perform the second-step reaction.

[0143] <Preparation of Plasmid Vector> To efficiently express PAL derived from Anabaena variabilis or Plagiochasma appendiculatum in E. coli, modified nucleotide sequences were designed taking into account the codon usage in E. coli. DNAs consisting of these modified nucleotide sequences were then chemically synthesized according to standard methods, and plasmid vectors (various PAL vectors) capable of expressing the various genes in E. coli were prepared in the same manner as described above.

[0144] Next, DNA amplified by PCR from FDC vector 1 and pTrcHis B vector (Thermo Fisher Scientific) was ligated to pZA33luc vector (Expressys) using the Gibson Assembly method to prepare an FDC plasmid vector (FDC vector 2) for measuring enzyme activity in E. coli.

[0145] <Preparation of medium for enzyme activity measurement and measurement of enzyme activity> The prepared vectors (5 μg of PAL vector, 5 μg of UbiX vector, and 5 μg of FDC vector 2) were introduced into Escherichia coli C41(DE3) strain (Lucigen Corporation, 100 μL) by the heat shock method to prepare transformants co-expressing various PALs, wild-type FDC, and UbiX.

[0146] These transformants were then cultured for 6 hours in LB medium supplemented with ampicillin, kanamycin, and chloramphenicol. The growth of these transformants reached a plateau after this 6-hour culture (preculture). Therefore, the bacterial mass at the start of the enzymatic reaction described below was uniform among the transformants.

[0147] In addition, a medium for measuring enzyme activity was prepared by adding 12 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, 20 g / L lactose, 100 mg / L ampicillin, 50 mg / L kanamycin, and 30 mg / L chloramphenicol, as well as 1 mM phenylalanine or 1 mM allylglycine as a substrate.

[0148] Then, 50 μL of the culture solution of the transformant and 1 mL of the medium for measuring enzyme activity were added to a 10 mL vial for HS / GSMS, and immediately thereafter, the vial was capped and the enzyme reaction was carried out at 37°C with a shaking speed of 180 rpm. 18 hours after the start of the reaction, the peak areas representing the amounts of styrene or 1,3-butadiene produced in the headspace of the vial were measured by GC-MS.

[0149] As a result, as shown in Figure 1, when phenylalanine was added as a substrate, styrene was produced for all PALs, as shown below. Furthermore, there was no significant difference in the amount of styrene produced among the three PALs.

[0150] On the other hand, when allylglycine was added as a substrate, butadiene was produced from both PALs, as shown in Figure 2. However, there were significant differences in the amount of butadiene produced, with the greatest butadiene production observed when AvPAL was used.

[0151] Example 3 As shown in Example 2, wild-type AvPAL was found to have high catalytic activity for the production of butadiene. Therefore, in order to further improve this high catalytic activity, mutations were introduced to substitute amino acids at the enzyme active site with other amino acids. The catalytic activity of the resulting mutants was then evaluated.

[0152] Specifically, primers encoding the amino acid sequence into which each mutation was introduced were designed and synthesized. Then, using the vector encoding wild-type AvPAL prepared in Example 2 as a template and the primers, a plasmid vector (PAL variant vector) capable of expressing PAL into which each mutation was introduced in Escherichia coli was prepared according to the Gibson Assembly protocol. Then, in the above <Preparation of medium for enzyme activity measurement and measurement of enzyme activity>, the PAL variant vector was introduced instead of the wild-type PAL vector, a transformant was prepared, and the enzyme activity was measured. The amount of butadiene produced when wild-type PAL was used is set to 1.0, and the amount of butadiene produced when the PAL variants were used is shown in Table 3 below.

[0153] [Table 3]

[0154] As shown in Table 3, as a result of introducing various amino acid substitutions, the following amino acid substitutions were found to improve butadiene production compared to the wild type before the introduction. (1) leucine at position 108 is replaced with methionine, phenylalanine, or valine; (2) Phenylalanine at position 107 is replaced by tryptophan; (3) Leucine at position 219 is replaced with isoleucine. (4) Asparagine at position 223 is replaced with isoleucine. (5) Leucine at position 104 is replaced with alanine. [Industrial Applicability]

[0155] As described above, the present invention provides a method for producing an unsaturated compound having at least two carbon-carbon double bonds using an enzyme. For example, the present invention makes it possible to produce butadiene using relatively inexpensive L-lysine as a starting material. Furthermore, the present invention enables the production of unsaturated compounds by biosynthesis rather than chemical synthesis, thereby reducing the burden on the environment. Therefore, the present invention is extremely useful in producing butadiene, a raw material for various synthetic polymers such as synthetic rubber.

Claims

1. A method for producing a chain unsaturated carboxylic acid compound represented by the following formula (3) or a geometric isomer thereof, the method comprising the step of eliminating a first amino group from a chain unsaturated carboxylic acid compound represented by the following formula (2) or a geometric isomer thereof, the chain having a first amino group and a first carbon-carbon double bond at a terminal, in the presence of phenylalanine ammonia-lyase, to form a second carbon-carbon double bond: 【Chemistry 1】 [In the above formula, (A) represents a linear hydrocarbon group having 0 to 5 carbon atoms which may be substituted, and when the carbon atom number is 2 to 5, a double bond may be formed between adjacent carbon atoms. R １ and R ２ 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.

2. The method according to claim 1, comprising the step of producing a chain unsaturated carboxylic acid compound represented by the following formula (2) or a geometric isomer thereof by eliminating a second amino group and a methylene group from a chain carboxylic acid compound represented by the following formula (1) having a first amino group and a second amino group at a terminal, or a geometric isomer thereof, in the presence of terminal alkene synthase BesC, and producing a chain unsaturated carboxylic acid compound represented by the formula (3) or a geometric isomer thereof from the compound or the geometric isomer thereof: 【Chemistry 2】 [In the above formula, (A) represents a linear hydrocarbon group having 0 to 5 carbon atoms which may be substituted, and when the carbon atom number is 2 to 5, a double bond may be formed between adjacent carbon atoms. R １ and R ２ 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.

3. A method for producing a chain unsaturated hydrocarbon compound represented by the following formula (4) or a geometric isomer thereof, comprising the steps of producing a chain unsaturated carboxylic acid compound represented by the formula (3) or a geometric isomer thereof by the method according to claim 1 or 2, and eliminating a carboxyl group from the unsaturated carboxylic acid compound or the geometric isomer thereof in the presence of ferulic acid decarboxylase: 【Transformation 3】 [In the above formula, (A) represents a linear hydrocarbon group having 0 to 5 carbon atoms which may be substituted, and when the carbon atom number is 2 to 5, a double bond may be formed between adjacent carbon atoms. R １ and R ２ 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.

4. The method according to any one of claims 1 to 3, wherein the phenylalanine ammonia-lyase has at least one of the following characteristics (1) to (5): (1) The amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is methionine, phenylalanine, or valine; (2) The amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is tryptophan. (3) The amino acid at position 219 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is isoleucine. (4) The amino acid at position 223 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is isoleucine. (5) The amino acid at position 104 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is alanine.

5. The present invention includes a phenylalanine ammonia-lyase variant into which at least one of the following amino acid substitutions (1) to (5) has been introduced, a DNA encoding the phenylalanine ammonia-lyase variant, or a vector into which the DNA has been inserted: An agent for promoting the production of a chain unsaturated carboxylic acid compound represented by the following formula (3) or a geometric isomer thereof from a chain unsaturated carboxylic acid compound represented by the following formula (2) having a first amino group and a first carbon-carbon double bond at a terminal thereof or a geometric isomer thereof: (1) The amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with methionine, phenylalanine, or valine; (2) the amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with tryptophan; (3) The amino acid at position 219 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with isoleucine; (4) The amino acid at position 223 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with isoleucine; (5) the amino acid at position 104 of the amino acid sequence set forth in SEQ ID NO: 2 or a corresponding position thereto is substituted with alanine; 【Chemistry 4】 [In the above formula, (A) represents an optionally substituted linear hydrocarbon group having 0 to 5 carbon atoms, and when the group has 2 to 5 carbon atoms, a double bond may be formed between adjacent carbon atoms. R 1 and R 2 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].

6. A terminal alkene-synthesizing enzyme BesC, DNA encoding the enzyme, or a vector into which the DNA has been inserted; The present invention comprises a phenylalanine ammonia-lyase variant into which at least one of the following amino acid substitutions (1) to (5) has been introduced, a DNA encoding the phenylalanine ammonia-lyase variant, or a vector into which the DNA has been inserted: An agent for promoting the production of a chain unsaturated carboxylic acid compound represented by the following formula (3) or a geometric isomer thereof from a chain carboxylic acid compound represented by the following formula (1) having a first amino group and a second amino group at a terminal thereof or a geometric isomer thereof: (1) The amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with methionine, phenylalanine, or valine; (2) the amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with tryptophan; (3) The amino acid at position 219 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with isoleucine; (4) The amino acid at position 223 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with isoleucine; (5) the amino acid at position 104 of the amino acid sequence set forth in SEQ ID NO: 2 or a corresponding position thereto is substituted with alanine; 【Transformation 5】 [In the above formula, (A) represents an optionally substituted linear hydrocarbon group having 0 to 5 carbon atoms, and when the group has 2 to 5 carbon atoms, a double bond may be formed between adjacent carbon atoms. R 1 and R 2 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].

7. A phenylalanine ammonia-lyase variant into which at least one of the following amino acid substitutions (1) to (5) has been introduced, DNA encoding the phenylalanine ammonia-lyase variant, or a vector into which the DNA has been inserted: ferulic acid decarboxylase, DNA encoding the ferulic acid decarboxylase, or a vector into which the DNA has been inserted, An agent for promoting the production of a chain unsaturated hydrocarbon compound represented by the following formula (4) or a geometric isomer thereof from a chain unsaturated carboxylic acid compound represented by the following formula (2) having a first amino group and a first carbon-carbon double bond at a terminal thereof or a geometric isomer thereof: (1) The amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with methionine, phenylalanine, or valine; (2) the amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with tryptophan; (3) The amino acid at position 219 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with isoleucine; (4) The amino acid at position 223 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with isoleucine; (5) the amino acid at position 104 of the amino acid sequence set forth in SEQ ID NO: 2 or a corresponding position thereto is substituted with alanine; 【Transformation 6】 [In the above formula, (A) represents an optionally substituted linear hydrocarbon group having 0 to 5 carbon atoms, and when the group has 2 to 5 carbon atoms, a double bond may be formed between adjacent carbon atoms. R 1 and R 2 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].

8. A terminal alkene-synthesizing enzyme BesC, DNA encoding the enzyme, or a vector into which the DNA has been inserted; A phenylalanine ammonia-lyase variant into which at least one of the following amino acid substitutions (1) to (5) has been introduced, DNA encoding the phenylalanine ammonia-lyase variant, or a vector into which the DNA has been inserted: ferulic acid decarboxylase, DNA encoding the ferulic acid decarboxylase, or a vector into which the DNA has been inserted, An agent for promoting the production of a chain unsaturated hydrocarbon compound represented by the following formula (4) or a geometric isomer thereof from a chain carboxylic acid compound represented by the following formula (1) having a first amino group and a second amino group at a terminal thereof or a geometric isomer thereof: (1) The amino acid at position 108 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with methionine, phenylalanine, or valine; (2) the amino acid at position 107 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with tryptophan; (3) The amino acid at position 219 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with isoleucine; (4) The amino acid at position 223 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to said position is substituted with isoleucine; (5) the amino acid at position 104 of the amino acid sequence set forth in SEQ ID NO: 2 or a corresponding position thereto is substituted with alanine; 【Transformation 7】 [In the above formula, (A) represents an optionally substituted linear hydrocarbon group having 0 to 5 carbon atoms, and when the group has 2 to 5 carbon atoms, a double bond may be formed between adjacent carbon atoms. R 1 and R 2 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].

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