JP2003225092A - Method for producing l-amino acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for readily and efficiently producing an L-amino acid having high optical purity and a new method for readily and efficiently producing an L-amino acid having high optical purity using a cinnamic acid derivative having a substituent on the phenyl group and an acrylic acid derivative having a heterocycle as a raw material. <P>SOLUTION: Remarkably high phenylalanine derivative-forming activity is found in a phenylalanine ammonia-lyase derived from plants, compared with a conventionally known enzyme derived from microorganisms. Examination results of the activity for forming not only the phenylalanine derivative but also industrially useful amino acids based on the wide substrate specificity of the phenylalanine ammonia-lyase derived from plants allow to solve the problems by using the enzyme. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a process for producing a corresponding optically active amino acid by adding ammonia to an acrylic acid derivative by the action of a plant enzyme. Optically active amino acids are useful as raw materials for the synthesis of pharmaceuticals, agricultural chemicals, and other fine chemicals.

[0002]

2. Description of the Related Art As a method for obtaining an optically active amino acid,
Many organic synthetic reactions utilizing asymmetric catalysts and fermentative production methods utilizing the specificity of microorganisms have been investigated.

As one of the production methods for obtaining optically active phenylalanine, a method for optically obtaining optically active phenylalanine by allowing phenylalanine ammonia lyase derived from a microorganism or a microorganism having the enzyme activity to act on cinnamic acid is described in British Patent No. 1489468, JP-A-53
Many reports have been made, such as Japanese Patent Publication No. -96388. Under high ammonia concentration, these acted by the enzyme to produce α-
It utilizes the reaction of adding an amino group to carbon,
L-phenylalanine can be produced with good optical purity.

Optically active L having a substituent on the phenyl group
As a method of obtaining a -phenylalanine derivative, a method of introducing a substituent on the phenyl group by using various modification reactions for L-phenylalanine can be considered. However, in such a method, a decrease in yield due to a side reaction of a portion other than the phenyl group and a decrease in optical purity due to progress of racemization under severe reaction conditions cannot be avoided. In addition, it is also difficult to obtain the regiospecificity of the substituent to be introduced on the phenyl group, resulting in low yield and difficulty in separation and purification, which is not practical.

Therefore, it is possible to use the above-mentioned phenylalanine ammonia-lyase to act the enzyme on a cinnamic acid derivative having a substituted phenyl group in which a desired substituent is introduced in advance. Since the enzymatic reaction proceeds under mild conditions, it is expected that the phenyl group and the substituents on the phenyl group will not be adversely affected by side reactions and that the optically active L-phenylalanine derivative can be obtained in high purity.

However, the substrate specificity of phenylalanine ammonia-lyase derived from microorganisms is generally strict, and the reactivity with substituted phenyl compounds is significantly lower than that of the original reaction of cinnamic acid to L-phenylalanine, or almost no reaction. Often not. For example,
Starting from a cinnamic acid derivative having a fluorinated phenyl group,
Method for obtaining optically active fluorinated phenylalanine by the action of the enzyme (JP-A-63-148992), and Rhodotoru
Only a few cases have been disclosed such as a method for obtaining a phenylalanine derivative using a specific microorganism of the genus la (US Pat. No. 5981239), applicable compounds are limited, and its productivity is also industrial. It was not enough for me.

Phenylalanine ammonia lyase is generally known to be distributed throughout the living world of animals, plants and microorganisms. That is, in microorganisms, Rhodotorula ru
bra (Japanese Patent Laid-Open No. 61-043993, GB1489468), Rhodospor
idium toruloides (JP-A-60-227670), Cladosp
oridium cladosporioides (Japanese Patent Laid-Open No. 62-111687), and many other reports.

On the other hand, in plants, Arabidopsis ( Arabidop
sis thaliana ), tea ( Camellia sinensis ), chickpea ( Cicer arietinum ), lemon ( Citrus limoni )
s ), cucumber ( Cucumis sativus L.), carrot ( Daucu
s carota ), purple ( Lithospermum erythrorhizo )
n ), tomato ( Lycopersicon esculentum ), tobacco ( N
icotiana tabacum ), rice ( Oryza sativa ), parsley ( Petroselinum crispum , Petroselinum hortense ),
Pinus taeda , Populus ( Populus kitakam )
iensis etc.), cherries ( Prunus avium ), raspberries ( Rubus idaeus ), eggplants ( Solanum tuberosum ), ( St
ylosanthes humilis ), Shaji soup ( Trifolium subt
errane ), grapes ( Vitis vinifera ), common beans ( Phaseolus vulgaris ) and the like are known.

Many phenylalanine ammonia lyase structural genes pal derived from plants (hereinafter referred to as "pal gene") have also been reported. That is, rice ( Oryza sativa , Biochim . Bio
phys . Acta (1993), 1171 (3), 321-322, Plant Mol . Bi
ol . (1995), 29 (3), 535-550), Cha ( Camellia sinen
sis , Theor . Appl . Genet . (1994), 89 (6), 671-67
5), Arabidopsis thaliana , Plant M
ol . Biol . (1995), 27, 327-338), purple ( Lithospe )
rmum erythrorhizon , Biosci . Biotech . Biochem . (199
7), 61 (12), 1995-2003), tomato ( Lycopersicon escul
entum , J. Biol . Chem . (1992), 267, 11824-11830), Shajisoukou ( Trifolium subterraneum , Gene (1994), 1
38 (1-2), 87-92), Peas ( Pisum sativum , Pla
nt Mol . Biol . (1992), 20 (1), 167-170, JP-A-5-1539
No. 78), Poplar ( Populus trichocarpa , Plant Phy
siol. (1993), 102, 71-83), tobacco (Nicotiana tabac
um , Plant Mol . Biol . (1996), 30, 711-722), soybean ( Glycine max , DNA Sequence (1991), 1 (5), 335-34.
6), sweet potato ( Ipomoea batatas , Plant Physiol .
(1989), 90 (4), 1403-1407), wheat ( Triticum aestivu
m ), parsley ( Petroselinum crispum ), sunflower ( Hel
ianthus annuus ), alfalfa ( Medicago sativ )
a ), etc., and the number of isomers and partial sequences existing in plants of the same species are clarified, the number is 40 or more.

[0010] Plant-derived phenylalanine ammonia-lyase has many conserved sequences with each other, and even if the amino acid sequence is the lowest, it shows 50% or more homology, and it is speculated that there are many commonalities in their functions and properties. it can.

Phenylalanine ammonia lyase derived from a plant is the first and the rate-determining reaction of the phenylpropanoid-isoflavonoid synthetic pathway in the secondary metabolism of plants, that is, an enzyme that catalyzes the deammonification reaction of phenylalanine. Known enzymes and their functions as genes involved in plant stress tolerance have been studied in detail. In addition, an attempt to express disease-resistant plants by expressing the pal gene in plants has been
Widely tried across applications.

[0012] Despite the fact that there are many reports as described above, unlike the enzyme derived from microorganisms, research from the viewpoint of substance production was carried out using some useful substances of the phenylpropanoid / isoflavonoid system. Although attempts have been made to increase the content ( Planta . (1979), 14 (6), 369-37
6., Biochem . Biophys . Acta . (1979), 563, 278-29
2.), Attempts to produce organic compounds that are general industrial materials are not known. Of course, no attempt has been reported to produce a substance by using a reverse reaction, an amino group addition reaction. In the first place, the plant-derived enzyme is not limited to this enzyme,
From the belief that it is less practical than microbial enzymes in terms of low stability, high substrate specificity, etc., there are very few examples used for substance production, and those skilled in the art can use organic compounds similar to those of the present invention. When devising a method for producing the L-phenylalanine derivative in (1), it was one of the most notable materials as long as an enzyme having the same catalytic function exists in another source.

[0013]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel method for obtaining an L-amino acid having a high optical purity, which is simple and efficient, and is cinnamic acid having a substituent on the phenyl group. It is an object of the invention to provide a novel method for easily and efficiently obtaining an L-amino acid having high optical purity by using an acrylic acid derivative such as a derivative as a raw material.

[0014]

[Means for Solving the Problems] The present inventors have paid attention to the broad substrate specificity of phenylalanine ammonia lyase derived from plants and examined the production activity of various industrially useful amino acids.

First, an ammonia lyase having higher reactivity with a cinnamic acid derivative having a substituent on the phenyl group was searched for in the entire living world of animals, plants and microorganisms.

As a result, the present inventors have found that these plant-derived phenylalanine ammonia lyases have a significantly higher phenylalanine derivative-forming activity than the conventionally known microorganism-derived enzyme. As described above, the formation process of phenylpropanoids, isoflavonoids, etc. in plants involved in these enzymes has been studied in detail, but this is the formation of cinnamic acid derivatives by the elimination of amino group of phenylalanine. The reverse reaction, that is, the possibility of addition of an amino group to a cinnamic acid derivative by these plant enzymes and the possibility of forming a phenylalanine derivative has not been investigated.

Furthermore, focusing on the broad substrate specificity of these plant-derived phenylalanine ammonia lyases, as a result of examining not only the phenylalanine derivative but also various industrially useful amino acid producing activities, these enzymes were
Using an acrylic acid derivative having a substituent and an aromatic ring structure which may contain a hetero atom as a substrate, the corresponding L-
The activity of producing amino acids was found.

As a result of further intensive studies, the present inventors have found that amino acids can be produced with extremely high efficiency by using a microorganism that highly expresses the plant-derived pal gene, and the present invention is completed. I arrived.

Conventionally, in plant-derived phenylalanine ammonia-lyase, the ability to catalyze the reaction to obtain an amino acid using an acrylic acid derivative having the structure represented by the above formula (1) as a substrate has not been known, and the inventors have found that This is a newly obtained finding.

In plant-derived phenylalanine ammonia-lyase, the ability to catalyze the reaction for obtaining a phenylalanine derivative using a cinnamic acid derivative represented by the above formula (3) having various substituents on the phenyl group as a substrate is known. This is a finding newly obtained by the inventors.

In the plant-derived phenylalanine ammonia lyase, the reaction of obtaining an L-amino acid is catalyzed by using the acrylic acid derivative having a heterocyclic substituent represented by the above formula (4) or (5) as a substrate. The ability is unknown, and is a finding newly obtained by the inventors.

That is, the present invention relates to the following [1] to [3]
0]. [1] The following formula (1)

[0023]

[Chemical 6]

(Wherein Z represents an aromatic ring group which may contain a hetero atom, R represents a substituent on the aromatic ring, and n
Represents an integer of 0 or more, and when n is 2 or more, R may be the same or different. ) A plant-derived phenylalanine ammonia lyase is allowed to act on the acrylic acid derivative represented by the formula (1) in the following formula (2):

[0025]

[Chemical 7]

(In the formula, Z represents an aromatic ring group optionally containing a hetero atom, R represents a substituent on the aromatic ring, and n
Represents an integer of 0 or more, and when n is 2 or more, R may be the same or different. The manufacturing method of L-amino acid shown by these. [2] R _n -Z- is the following formula (3)

[0027]

[Chemical 8]

(However, R is a substituent on the benzene ring, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a fluorine atom, a chlorine atom, a bromine atom, an amino group, a nitro group,
It represents a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. n represents an integer of 0 to 5, and when n is 2 or more, R may be the same or different. ) The method for producing an L-amino acid according to the above [1]. [3] R _n -Z- is the following formula (4)

[0029]

[Chemical 9]

(Where X is S, O, NH or NR
¹ , R ¹ represents an alkyl group having 1 to 6 carbon atoms, R is a substituent on the heterocycle, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a fluorine atom, a chlorine atom, a bromine atom, an amino group. Represents a group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. n represents an integer of 0 to 3, and when n is 2 or more,
R may be the same or different. ) The method for producing an L-amino acid according to the above [1]. [4] R _n -Z- is the following formula (5)

[0031]

[Chemical 10]

(Where X is S, O, NH or NR
¹ , R ¹ represents an alkyl group having 1 to 6 carbon atoms, R is a substituent on the heterocycle, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a fluorine atom, a chlorine atom, a bromine atom, an amino group. Represents a group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. n represents an integer of 0 to 3, and when n is 2 or more,
R may be the same or different. ) The method for producing an L-amino acid according to the above [1].

[5] The method for producing an L-amino acid according to any one of the above [1] to [4], which comprises using a plant cultured cell containing phenylalanine ammonia lyase and / or a treatment composition thereof. [6] The method for producing an L-amino acid according to any one of [1] to [4] above, which comprises using a plant tissue treatment composition containing phenylalanine ammonia lyase. [7] Any of the above-mentioned [1] to [4], wherein a plant-derived phenylalanine ammonia-lyase gene is present in a plant so that it can be expressed, and a transformed plant cultivated product or a processed product of the cultivated product is used. The method for producing an L-amino acid according to Crab.

[8] The plant-derived phenylalanine ammonia-lyase gene is present in a microorganism so that it can be expressed, and an enzyme obtained from the transformed microorganism culture, treated product or culture is used [1]. To the method of producing an L-amino acid according to any one of [4]. [9] The plant-derived phenylalanine ammonia lyase gene is pal2 derived from Lithospermumerythrorhizon.
The method for producing an L-amino acid according to the above [7] or [8], which is a gene encoding an amino acid sequence having 70% or more sequence homology with the amino acid sequence derived from the base sequence of the gene. [10] A plant-derived phenylalanine ammonia-lyase gene is pal-derived from Lithospermum erythrorhizon.
80% amino acid sequence derived from the nucleotide sequences of 2 genes
The method for producing an L-amino acid according to the above [7] or [8], which is a gene encoding an amino acid sequence having the above sequence homology.

[11] The plant from which the phenylalanine ammonia-lyase gene is derived is Lithospermum and / or
The method for producing an L-amino acid according to any one of [1] to [8] above, which belongs to the genus Camellia . [12] The method for producing an L-amino acid according to any of the above [8] to [11], wherein the microorganism is a bacterium, yeast and / or filamentous fungus. [13] L- according to the above [12], wherein the bacterium is Escherichia coli.
Amino acid manufacturing method.

[14] The method for producing an L-amino acid according to any one of the above [2], [5] to [13], wherein at least one R in the formula (3) is a hydroxyl group. [15] The method for producing an L-amino acid according to any one of the above [2], [5] to [13], wherein at least one of the substituents R in the formula (3) is a cyano group. [16] The above [2], [5] to [13], wherein at least one of the substituents R in the formula (3) is a carboxyl group.
The method for producing an L-amino acid according to any one of 1.

[17] The method for producing an L-amino acid according to any one of the above [2], [5] to [13], wherein at least one of the substituents R in the formula (3) is an amide group. [18] The method for producing an L-amino acid according to any one of the above [2], [5] to [13], wherein at least one of the substituents R in the formula (3) is a halogen group. [19] The method for producing an L-amino acid according to any one of [2] and [5] to [13] above, wherein at least one of the substituents R in the formula (3) is an amino group.

[20] The method for producing an L-amino acid according to any one of [2], [5] to [13] above, wherein at least one of the substituents R in the formula (3) is a nitro group. [21] The above [2], [5] to [1] wherein at least one of the substituents R in the formula (3) is a hydroxymethyl group.
[3] The method for producing an L-amino acid according to any one of [3]. [22] The method for producing an L-amino acid according to any one of [2] and [5] to [13] above, wherein at least one of the substituents R in the formula (3) is an alkyl group having 1 to 6 carbon atoms.

[23] The above-mentioned [2], [5] to [1] wherein the substituent R in the above formula (3) is any of a cyano group, a hydroxyl group, a nitro group or a carboxyl group and n is 1.
[3] The method for producing an L-amino acid according to any one of [3]. [24] The method for producing an L-amino acid according to any one of [2] and [5] to [13] above, wherein the L-amino acid is L-phenylalanine.

[25] L- according to any one of the above [3] and [5] to [13], wherein X in the above formula (4) is O.
Amino acid manufacturing method. [26] The above [3], wherein X in the formula (4) is S,
The method for producing an L-amino acid according to any of [5] to [13]. [27] The above [3], wherein X in the formula (4) is NH,
The method for producing an L-amino acid according to any of [5] to [13].

[28] The method for producing an L-amino acid according to any one of the above [4] to [13], wherein X in the formula (5) is O.

[29] The method for producing an L-amino acid according to any of the above [4] to [13], wherein X in the formula (5) is S. [30] The method for producing an L-amino acid according to any of the above [4] to [13], wherein X in the formula (5) is NH.

[0043]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The method for producing an L-amino acid of the present invention comprises an optically active amino acid from an acrylic acid derivative having an aromatic ring group which may have various substituents and may also contain a hetero atom,
It can be easily obtained by a one-step enzymatic reaction.

The method for producing L-amino acids of the present invention is that an optically active amino acid can be easily obtained from an acrylic acid derivative having a benzene ring group having various substituents by a one-step enzymatic reaction. is there.

Substituents introduced in advance into the benzene ring group such as cyano group, hydroxyl group, carboxyl group, amide group, halogen, amino group, nitro group, hydroxymethyl group, and alkyl group having 1 to 6 carbon atoms are used in this reaction. It is retained until the end of the reaction without being affected under mild conditions of
Approximately 10 corresponding L-phenylalanine derivatives were obtained.
A high yield can be obtained with a conversion rate of 0%.

Further, according to the method for producing an L-amino acid of the present invention, an optically active amino acid having a heterocyclic structure can be easily obtained from an acrylic acid derivative having a heterocyclic structure by a one-step enzymatic reaction.

The heterocyclic structure such as furyl group, thienyl group and pyrrole group in the acrylic acid derivative is not affected under the mild conditions of this reaction until the end of the reaction, and the corresponding desired L- Amino acids can be obtained in good yield at a conversion rate of almost 100%.

The acrylic acid derivative having various substituents, which is a reaction raw material of the present invention, is a method by a so-called Parkin reaction in which an aldehyde group of an aldehyde derivative having an arbitrary substituent is reacted with acetic anhydride ( Arch). . Phar
m . (Weinheim) (1994), 327 (10), 619-625, etc.),
Alternatively, a method of reacting malonic acid or the like in the presence of piperidine in a pyridine solvent ( J Chem . Soc . (1939), 357-360
Etc.) and various other amendments ( Synth . Commun . (1999),
29 (4), 573-581 etc.) and the like.

There is no particular limitation on the plant that provides the phenylalanine ammonia lyase activity applied in the present invention. Specifically, as described above, Arabidopsis thaliana , tea ( Camellia sinensi )
s ), chickpea ( Cicer arietinum ), lemon ( Citru
s Limonis ), cucumber ( Cucumis sativus L.), carrot ( Daucus carota ), purple ( Lithospermum erythr )
orhizon ), tomato ( Lycopersicon esculentum ), tobacco ( Nicotiana tabacum ), rice ( Oryza sativa ), parsley ( Petroselinum crispum ), Petroselinum hortens
e ), Pinus taeda ( Pinus taeda ), Poplar ( Populus k )
itakamiensis etc.), cherries ( Prunus avium ), raspberries ( Rubus idaeus ), eggplants ( Solanum tuberosu )
m ), Stylos ( Stylosanthes humilis ), Shajisou ( Trifolium subterrane ), grapes ( Vitis vinife )
ra ), common beans ( Phaseolus vulgaris ), etc. are known, but they are enzymes that are almost universally present in plants.

As one of the embodiments of the present invention, phenylalanine ammonia lyase isolated from plants is used. That is, a known method for separating and purifying an enzyme from a plant, for example, a reaction catalyst for the enzyme separated and purified by an ordinary method such as acetone fractionation precipitation, ammonium sulfate precipitation, various separation columns, etc., from a ground plant extract Can be used as Specific methods for separating and purifying phenylalanine ammonia lyase from various plants include J. Koukol.
( J Biol . Chem . (1961), 236 (10), 2692-269)
8), reported by EA Havir et al. ( Biochemistry (1973), 12,
1583-) etc. are known.

Further, as one of different embodiments of the present invention, the treated product of the plant as described above is subjected to a target reaction. Examples of processed plants include milled products of plants.
Lyophilized products and extracts, those obtained by concentrating and extracting the components that catalyze the desired reaction from the extracts, and those obtained by treating these plant products and extracts, and the extracted components being immobilized on a sparingly soluble carrier, Etc. can be used for the reaction.

Examples of such an immobilization carrier include polyacrylic acid, polymethacrylic acid, polyacrylamide, polyvinyl alcohol, poly-N-vinylformamide, polyallylamine, polyethyleneimine, methylcellulose, glucomannan, alginate, carrageenan, etc.
Further, compounds capable of forming a poorly water-soluble solid content including a plant extract component, such as these (crosslinked) polymers, can be used alone or in combination.

As one of the different embodiments of the present invention, a cultured cell of the plant is used. That is, a general method for obtaining cultured cells of plants, for example, in the presence of plant hormones such as indoleacetic acid and kinetin, Murashige & Skoo
g Culture is performed in various known plant cell culture media selected according to the type of plant such as complete medium, LS medium, M9 medium, Gamborg B-5 medium, and the obtained cultured cells are subjected to the reaction. At this time, medium / culture conditions modified to enhance phenylalanine ammonia-lyase activity may be used.

[0054] For example, as culture conditions for obtaining cultured cells in which the enzyme activity of Lithospermum erythrorhizon is induced,
Report by Yazaki et al. ( Biosci . Biotech . Biochem . (1997),
61 (12), 1995-2003), and Klaus et al. 'S report on the relationship between the enzyme activity and culture conditions in cultured Petroselinum hortense cells ( Archiv . Biochem . Biophis . (197
5), 66, 54-62) etc. are known. In addition, a milled product / lyophilized product or an extract of the plant cultured cells thus obtained, which is obtained by concentrating / extracting a component that catalyzes a target reaction from the extract, further, an extract of the plant cultured cells, The extract components immobilized on a sparingly soluble carrier can be used in the reaction.

Furthermore, as one of the different embodiments of the present invention, the pal gene derived from a plant can be introduced into a microorganism or plant so that it can be expressed and the transformed microorganism or transformed plant can be used. The host organism used for gene expression is not particularly limited as long as a method for incorporating and expressing a foreign gene is known. Such organisms include, for example, the genus Escherichia , Pseudomon
Bacteria such as as and Bacillus , Saccharomyces , Schiz
Yeasts such as the genus osaccharomyces and filamentous fungi such as the genus Aspergillus can be used.

The pal gene used in the present invention may be a naturally occurring pal gene or a gene having the same function (encoding a mutant enzyme having the same activity), for example, pal derived from Lithospermum erythrorhizon.
2 genes (database accession No. D83076),
Alternatively, pal encoding an amino acid sequence having 70% or more sequence homology with the amino acid sequence derived from the base sequence of the pal2 gene derived from Lithospermum erythrorhizon
Examples of the gene include a pal gene encoding an amino acid sequence having a sequence homology of 80% or more with an amino acid sequence derived from the nucleotide sequence of the pal2 gene derived from Lithospermum erythrorhizon . Since the sequence homology of the plant pal gene is sufficiently high and the functions performed in the plant are the same, there are many commonalities in functions and properties, and the same effect can be expected in this production method, as described above. On the street.

For isolation of the enzyme gene cDNA from a plant, preparation of a transformed microorganism using the gene, and method of fractionating the enzyme from the transformed microorganism, for example, Lithospermum erythrorhizon was used. As an example, the report of Yazaki et al. ( Biosci . Biotech . Biochem . (199
7), 61 (12), 1995-2003), also Petroselinum crispum
Report of W. Schulz et al. ( FEBS Letter
(1989), 258 (2), 335-338) and many others.

Next, an example of a method for obtaining the pal gene, a method for preparing an expressible plasmid, and a method for introducing / expressing these plasmids into various organisms will be described in more detail. <Method of obtaining pal gene> The plant-derived phenylalanine ammonia lyase gene pal can be obtained from a cDNA library prepared by a known method using a partial fragment corresponding to a conserved sequence of phenylalanine ammonia lyase as a probe. First, in order to obtain the necessary cDNA, it is possible to isolate and purify a conventional mRNA and then use this as a template, and obtain it using Reverse transcriptase, but in recent years, using a heat-resistant Reverse transcriptase,
A sufficient amount of c can be easily prepared by PCR using total RNA or mRNA as a template.
DNA can be obtained. The cDNA thus obtained is ligated to an appropriate plasmid vector such as pBR322 or pUC18 to transform E. coli as a host.

The desired pal gene can be obtained from the cDNA library prepared as described above by a colony hybridization method using an oligo DNA encoding a conserved sequence of phenylalanine ammonia-lyase as a probe. As described above, the plant-derived phenylalanine ammonia lyase has highly homologous sequences that are presumed to be conserved sequences throughout the sequence, and a position suitable for the purpose is selected to prepare an oligo DNA serving as a probe, which is used. be able to.

The position and direction of the pal gene inserted in the plasmid can be confirmed by preparing a plasmid from the positive candidate transformant obtained by colony hybridization and performing PCR using appropriate primers in combination. . Since the primary sequences of many plant-derived pal genes are published on databases such as GenBank and EMBL, the position / direction can be confirmed by creating a restriction enzyme map for those for which sequence information exists.

<Method for producing expression plasmid and method for producing recombinant microorganism used for production> The pal gene is bacterium such as Escherichia coli by a suitable vector (a specific example will be described later); Saccha
By introducing into a microorganism such as yeast such as romyces cerevisiae ; preferably a bacterium such as Escherichia coli and expressing it, a recombinant having the desired phenylalanine derivative-forming activity can be obtained. The phenylalanine derivative produced is
As described below, the microbial reaction product can be isolated and purified by a usual method for separating the reaction product from the reaction product.

The method for producing a plasmid containing a foreign gene and the procedure or method for introducing and expressing the plasmid into a microorganism such as Escherichia coli are the methods or methods conventionally used in the field of genetic engineering (for example, "Vectors for cloning"). g
enes ", Methods in Enzymology , 216, p. 469-631, 199
2, Academic Press, and "Other bacterial syste
ms ", Methods in Enzymology , 204, p.305-636, 199 1,
Academic Press) (see Academic Press).

Foreign gene to E. coli (pal gene group)
There are several established efficient methods such as Hanahan's method and rubidium method, which can be used (for example, Sambrook, J., Fritsch, EF,
Maniatis, T., "Molecular Cloning -A laboratory ma
nual. "Cold Spring Harbor Laboratory Press, 1989.) Expression of a foreign gene in E. coli may be performed according to a standard method (for example," Molecular cloning -A lab "described above).
oratory manual. "), pUC, pBluescript, etc.
It can be carried out using an E. coli vector having a lac promoter or the like. The present inventors inserted the pal gene downstream of the lac promoter using the E. coli vector pBluescript II SK- having a lac promoter and expressed it in E. coli.

Pal to yeast Saccharomyces cerevisiae
There are already established methods such as the lithium method for gene introduction, and these methods may be used (for example, refer to “New Biotechnology of Yeast” published by The Medical Publication Center edited by Yuichi Akiyama, edited by Bioindustry Association). For expression of a foreign gene in yeast, a promoter and terminator such as PGK or GPD (GAP) is used to construct an expression cassette in which the foreign gene is inserted between the promoter and the terminator so as to be transcriptionally regulated. Replace the cassette with S. cerevisiae vector, for example, YRp system (a yeast multicopy vector whose origin of replication is the ARS sequence of the yeast chromosome), YE
It can be carried out by inserting into a vector such as p-type (a yeast multicopy vector having an origin of replication of 2 μm DNA of yeast), YIp-type (a vector for integrating yeast chromosome that does not have an origin of replication of yeast) (the above-mentioned. See "New Biotechnology for Yeasts" published by the Medical Publishing Center, ABC Series "Genetic Engineering for Material Production" published by Asakura Shoten, Japan Society of Agricultural Chemistry).

<Cultivation Method of Recombinant Microorganisms> The transformant related to the present invention can be cultured according to a general culture method for host microorganisms. As a medium carbon source for culturing a transformed microorganism, those that can be used as a carbon source by the host microorganism, such as glucose and sucrose,
Sugars such as fructose and molasses, organic substances such as ethanol, acetic acid, citric acid, succinic acid, lactic acid, benzoic acid, and fatty acids, or their alkali metal salts, aliphatic hydrocarbons such as n-paraffin, and also peptone, meat, etc. Extracts, fish extracts, soybean flour, bran, malt extracts, potato extracts, and other natural organic substances, alone or in combination, are usually 0.01% to 30%, preferably 0.1%.
It can be used at a concentration of about 10%. For example, when Escherichia coli is used as a transformant, glucose, peptone, meat extract, malt extract, etc. are preferably used.

The medium nitrogen source for culturing the microorganisms can be used by the host microorganism as a nitrogen source, for example, inorganic nitrogen compounds such as ammonium sulfate, ammonium phosphate, sodium nitrate and potassium nitrate, urea,
Nitrogen-containing organic substances such as uric acid, peptone, meat extract, fish extract, soybean flour, malt extract, potato extract and other natural organic substances, alone or in combination, are usually 0.01% to 30%, preferably 0%. It can be used at a concentration of about 1% to 10%. For example, when Escherichia coli is used as a transformant, ammonium sulfate, peptone, meat extract, malt extract, etc. are preferably used.

Further, if necessary, a phosphate such as potassium dihydrogen phosphate; a metal salt such as magnesium sulfate, ferrous sulfate, calcium acetate, manganese chloride, copper sulfate, zinc sulfate, cobalt sulfate and nickel sulfate. It is added to improve the growth of bacteria. The addition concentration varies depending on the culture conditions, but is usually 0.01% to 5% for phosphate, 10 ppm to 1% for magnesium salt, and about 0.1 ppm to 1000 ppm for other compounds. Further, depending on the medium to be selected, the growth of the bacterium can be improved by adding, for example, yeast extract, casamino acid, yeast nucleic acid in an amount of about 1 ppm to 100 ppm as a source of vitamins, amino acids, nucleic acids and the like.

If necessary, an inducer corresponding to the expression promoter of the vector can be added to the culture in order to highly express the enzyme gene used for transformation in the host microorganism. For example, when the Lac promoter is used, IPTG (isopropyl-1-thio-β-D
-Galactoside) is about 0.01 mM to 10 mM, and when a drug-resistant promoter such as ampicillin or kanamycin is used, the corresponding drug is 0.1 ppm to 1000 mM.
Add about ppm.

The pH of the culture is the same regardless of the composition used.
Is adjusted to 5-9, preferably 5.5-8. In addition, the microbial cells pre-cultured in the medium as described above are collected from the culture solution by a method such as centrifugation or membrane filtration and used for the reaction to reduce contaminants brought in from the culture solution. It is useful for simplifying the fractionation of the product.

The transformed microorganism obtained by culturing can be recovered as it is by a known method such as centrifugation or membrane separation, or the culture solution itself can be used for the desired reaction.
Furthermore, the transformed microorganism is crushed, frozen and dried, a cell-free extract from microbial cells, and a cell-free extract obtained by concentrating and extracting components that catalyze the desired reaction, and these transformed microorganisms. Bacterial cells and processed products thereof,
The extract, the extract components immobilized on a poorly soluble carrier, and the like can be subjected to the reaction.

The immobilizing carrier used for such purpose includes polyacrylic acid, polymethacrylic acid, polyacrylamide, polyvinyl alcohol, poly-N-vinylformamide, polyallylamine, polyethyleneimine,
Methyl cellulose, glucomannan, alginate, carrageenan, etc., and further (cross-linked) polymers thereof, etc., which are used alone or in combination to form a compound that forms a poorly water-soluble solid content in which microorganisms or their extract components are incorporated. You can In addition, activated carbon, porous ceramics,
It is also possible to use a substance such as a glass fiber, a porous polymer molded product, or a nitrocellulose membrane, which is preliminarily formed as a solid substance and holds the transformed microorganism or its extract or extract component.

<Method for Producing Recombinant Plant Used in Production and Culture Method> As described above, a plant producing a phenylalanine derivative can be produced by introducing the pal gene into the cell. As a preferred example, for example, by preparing a plasmid containing the above-mentioned pal gene, introducing this into a suitable plant such as tobacco, and expressing it,
A plant that produces the desired phenylalanine derivative can be obtained. Many research papers have been published on methods for introducing and expressing various genes in plants, and plants with increased target activity can be obtained by these known methods ( Plant Mol . Biol . (1999), 39 (4), 683-6
93, PlantBiotechnol . (Tokyo) (1998), 15 (4), 189-19
3, Plant Physiol . (1996), 112 (4), 1617-1624).

As a method for introducing a foreign gene into a plant, a method via a plant pathogenic bacterium Agrobacterium tumefaciens , an electroporation method, a method using a particle gun, etc. are known, and these methods are selected depending on the kind of the plant to be introduced. Can be used properly. The promoters include the 35S promoter of the cauliflower mosaic virus (CaMV), which is a systemic high expression promoter,
Those which are expressed in various organs can also be used.
The binary vector pBI121 containing the CaMV 35S promoter is available from Clontech,
It is widely used as a vector for mediating Agrobacterium tumefaciens . It has already been shown that the pal gene can be introduced into plants such as tobacco by using a particle gun, and that the pal gene is expressed and functions ( Shokubutsu Soshiki Baiyo (1995), 1).
2 (2), 165-171).

A method for producing a plasmid containing a foreign gene, a procedure and a method for introducing and expressing the plasmid into a plant (cells such as leaves, stems, roots) are not limited to those described in the present invention. It may be carried out in accordance with the method or method (for example, Isao Ishida, Norihiko Misawa, Introduction to Cell Engineering Experiment Operation, Kodansha, 1992) including those commonly used in the field of genetic engineering of plants. The recombinant plant prepared as described above can be prepared according to the general method for culturing plant cells as described above.

The L-amino acid producing reaction in the present invention is carried out by various known methods as described above, such as plant-derived phenylalanine ammonia lyase, a plant-treated product or plant cultured cells having the enzyme activity, or the plant. The enzyme produced by a microorganism transformed with the enzyme gene isolated from the above, or a transformed microbial cell and a treated product thereof, and further the enzyme produced by a plant transformed with the enzyme gene, Alternatively, in the presence of a transformed plant and a treated product thereof, an acrylic acid derivative as a reaction raw material is 1 ppm to 20%, preferably 10 ppm to 10%.
%, And ammonia is added so that the final concentration is 2 M to 12 M, and the pH is 8.5 to 11, preferably 9
It can be achieved by adjusting the reaction time to about 10.5 and reacting for about 1 to 200 hours.

As the acid used for adjusting the pH,
Inorganic acids such as sulfuric acid, hydrochloric acid, phosphoric acid, boric acid and carbonic acid, organic acids such as formic acid, acetic acid and propionic acid, and salts thereof can be used. At this time, it is useful to use a volatile acid because the reaction solution can be sterilized and distilled off to omit the step of desalting and to easily separate the product. Carbonic acid is suitable as such an acid. What is carbonic acid in this case?
As carbon dioxide, it contains carbonic acid which is dissolved in an aqueous solution by bubbling or the like and generated by dissociation. Further, salts of these acids and ammonia can be used as an ammonia source for the reaction liquid, and it is preferable to use ammonium carbonate or ammonium hydrogen carbonate as a part or all of the ammonia source for the reason as described above.

The acrylic acid derivative to be added does not necessarily have to be completely dissolved, but it is also possible to add a solvent, a surfactant or the like which improves the solubility and dispersibility in the reaction solution. . Further, the compound may be added continuously or intermittently depending on the consumption of the compound due to the progress of the reaction, and the concentration of the compound in the reaction solution in this case is not limited to the above.

The L-amino acid formed in the reaction solution may be a known method such as centrifugation, membrane filtration, vacuum drying, distillation, solvent extraction, salting out, ion exchange, various chromatography, etc., depending on the properties of the reaction solution. Is collected in. It can be achieved simply as follows. For example, filtration from the reaction solution,
After removing the enzyme, the enzyme-treated product, the transformed microorganism and its treated product by centrifugation, dialysis, or the like, solvent extraction or adjustment of the reaction solution to acidic to precipitate the unreacted starting acrylic acid derivative and remove the precipitate.

The pH of the obtained supernatant is again adjusted to the vicinity of the isoelectric point of the L-amino acid, and the precipitated derivative is similarly recovered. In this way, the product can be recovered from the reaction solution in good yield and purity. In addition, it is useful to remove excess ammonia from the reaction solution in advance by distillation or to distill off water to increase the concentration in order to improve the product recovery rate at the isoelectric point.

More simply, the pH of the reaction solution is adjusted with a volatile acid. When the conversion rate is sufficiently high, the product is converted to the ammonium salt of L-amino acid by sterilization as it is, and when a large amount of the raw material remains, acidification is performed by solvent extraction and then water / acid base is distilled off. It can be isolated. Carbonic acid and its ammonium salt are preferable as the volatile acid suitable for such a method.

Depending on the nature of the reaction product, the reaction rate may decrease due to the accumulation of the product in the reaction solution. In such a case, a method of adding water containing ammonia, physiological saline, and a reaction buffer solution to the reaction solution and continuously diluting the reaction solution according to the concentration of the product is preferable. When the reaction rate decreases, the bacteria are collected, the supernatant is recovered as a product solution, and the collected bacteria are returned to the solution or suspension containing the reaction raw materials to recover the reaction speed. You can These methods can be repeated as many times as long as the ammonia lyase activity of the microorganism is maintained.

The compound as the raw material of the present invention has the above formula (1)

[0083]

[Chemical 11]

(Wherein Z represents an aromatic ring group which may contain a hetero atom, R represents a substituent on the aromatic ring, and n
Represents an integer of 0 or more, and when n is 2 or more, R may be the same or different. ) Is an acrylic acid derivative. Further, the obtained compound has the above formula (2)

[0085]

[Chemical 12]

(Wherein Z represents an aromatic ring group which may contain a hetero atom, R represents a substituent on the aromatic ring, and n
Represents an integer of 0 or more, and when n is 2 or more, R may be the same or different. ) Is an L-amino acid.

In the above formula, Z is, for example, the following formulas (6) to (10).

[0088]

[Chemical 13]

The groups represented by the following formulas (11) to (15)

[0090]

[Chemical 14]

The groups represented by the following formulas (16) to (20)

[0092]

[Chemical 15]

The groups represented by the following formulas (21) to (25)

[0094]

[Chemical 16]

The groups represented by the following formulas (26) to (30)

[0096]

[Chemical 17]

The groups represented by the following formulas (31) to (34)

[0098]

[Chemical 18]

The groups represented by the following formulas (35) to (38)

[0100]

[Chemical 19]

The groups represented by the following formulas (39) to (42)

[0102]

[Chemical 20]

The groups represented by the following formulas (43) to (45)

[0104]

[Chemical 21]

A group represented by the following formulas and the following formulas (46) to (5)
0)

[0106]

[Chemical formula 22]

A group represented by the following formulas and the following formulas (51) to (5)
5)

[0108]

[Chemical formula 23]

A group represented by the following formulas and the following formulas (56) to (5)
8)

[0110]

[Chemical formula 24]

Examples include groups represented by:

R is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a fluorine atom, a chlorine atom, a bromine atom,
Amino group, nitro group, hydroxymethyl group, carbon number 1
Examples thereof include an alkyl group having 6 or an alkoxy group having 1 to 6 carbon atoms.

N can be an integer which is 0 or more and is equal to or less than the number of substitutable positions of the aromatic ring minus 1. When n is 2 or more, R may be the same or different.

Further, in the compound as the raw material of the present invention, in the above formula (1), R _n -Z- is represented by the following formula (3):

[0115]

[Chemical 25]

(Wherein R is a substituent on the benzene ring, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a fluorine atom, a chlorine atom, a bromine atom, an amino group, a nitro group,
It represents a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. n represents an integer of 0 to 5, and when n is 2 or more, R may be the same or different. ) Is a compound represented by.

Further, the obtained compound has the formula (2) wherein R _n -Z- is the formula (4) (wherein R is a substituent on the benzene ring, and a cyano group, a hydroxyl group, a carboxyl group, Amide group, fluorine atom, chlorine atom, bromine atom, amino group, nitro group, hydroxymethyl group, carbon number 1 to 6
Represents an alkyl group or an alkoxy group having 1 to 6 carbon atoms. n represents an integer of 0 to 5, and when n is 2 or more, R may be the same or different. ) Is a compound represented by.

The compound corresponding to the raw material of the formula (1) is R _n −
Specific examples of the compound in which Z- is represented by the formula (3) include 2-
Cyanocinnamic acid, 3-cyanocinnamic acid, 4-cyanocinnamic acid,
2-hydroxycinnamic acid, 3-hydroxycinnamic acid, 4-hydroxycinnamic acid, 3,4-dihydroxycinnamic acid, 2-nitrocinnamic acid, 3-nitrocinnamic acid, 4-nitrocinnamic acid, 2
-Carboxycinnamic acid, 3-carboxycinnamic acid, 4-carboxycinnamic acid, 2-aminocinnamic acid, 3-aminocinnamic acid,
4-aminocinnamic acid, 2-chlorocinnamic acid, 3-chlorocinnamic acid, 4-chlorocinnamic acid, 2-fluorocinnamic acid, 3-fluorocinnamic acid, 4-fluorocinnamic acid, 2-bromocinnamic acid,
3-bromocinnamic acid, 4-bromocinnamic acid, 2-iodocinnamic acid, 3-iodocinnamic acid, 4-iodocinnamic acid, 2-methylcinnamic acid, 3-methylcinnamic acid, 4-methylcinnamic acid, 2- Methoxycinnamic acid, 3-methoxycinnamic acid, 4-methoxycinnamic acid, 4-isopropylcinnamic acid, 4-t-butylcinnamic acid,
4-methoxy-3-methylcinnamic acid and the like can be mentioned.

Correspondingly produced amino acids are 2-cyano-L-phenylalanine, 3-cyano-L-phenylalanine, 4-cyano-L-phenylalanine, 2-hydroxy-L-phenylalanine and 3-hydroxy-L-.
Phenylalanine, 4-hydroxy-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine,
2-nitro-L-phenylalanine, 3-nitro-L-
Phenylalanine, 4-nitro-L-phenylalanine, 2-carboxy-L-phenylalanine, 3-carboxy-L-phenylalanine, 4-carboxy-L-
Phenylalanine, 2-amino-L-phenylalanine, 3-amino-L-phenylalanine, 4-amino-
L-phenylalanine, 2-chloro-L-phenylalanine, 3-chloro-L-phenylalanine, 4-chloro-L-phenylalanine, 2-fluoro-L-phenylalanine, 3-fluoro-L-phenylalanine, 4-
Fluoro-L-phenylalanine, 2-bromo-L-phenylalanine, 3-bromo-L-phenylalanine,
4-Bromo-L-phenylalanine, 2-iodo-L-
Phenylalanine, 3-iodo-L-phenylalanine, 4-iodo-L-phenylalanine, 2-methyl-
L-phenylalanine, 3-methyl-L-phenylalanine, 4-methyl-L-phenylalanine, 2-methoxy-L-phenylalanine, 3-methoxy-L-phenylalanine, 4-methoxy-L-phenylalanine, 4
-Isopropyl-L-phenylalanine, 4-t-butyl-L-phenylalanine, 4-methoxy-3-methyl-L-phenylalanine and the like.

For example, when the enzyme derived from Lithospermum erythrorhizon is used, 2-cyanocinnamic acid, 4-cyanocinnamic acid, 3-hydroxycinnamic acid, 4-hydroxycinnamic acid and 3,4-dihydroxycinnamic acid are suitable as raw materials. , 2-cyano-L-phenylalanine and 4-cyano-, respectively.
L-phenylalanine, 3-hydroxy-L-phenylalanine (metatyrosine), 4-hydroxy-L-phenylalanine (tyrosine), 3,4-dihydroxy-L
-Phenylalanine (DOPA) etc. are obtained.

The present invention is effectively applied to a cinnamic acid derivative having a substituent, that is, a compound in which n is 1 to 5 in the formula (3). However, the cinnamic acid derivative of the present invention contains cinnamic acid, Correspondingly, the phenylalanine derivative also includes phenylalanine.

In particular, the compound as the raw material of the present invention is
In the above formula (1), R _n -Z- is the following formula (4)

[0123]

[Chemical formula 26]

(In the formula, X is S, O, NH or NR.
¹ , R ¹ represents an alkyl group having 1 to 6 carbon atoms, R is a substituent on the heterocycle, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a fluorine atom, a chlorine atom, a bromine atom, an amino group. Represents a group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. n represents an integer of 0 to 3, and when n is 2 or more,
R may be the same or different. ), And the obtained compound is the compound of the formula (2) in which R _n -Z-
Is the above formula (4)

(In the formula, X is S, O, NH or NR.
¹ , R ¹ represents an alkyl group having 1 to 6 carbon atoms, R is a substituent on the heterocycle, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a fluorine atom, a chlorine atom, a bromine atom, an amino group. Represents a group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. n represents an integer of 0 to 3, and when n is 2 or more,
R may be the same or different. ) Is a compound represented by.

Further, in the compound as a raw material of the present invention, in the above formula (1), R _n -Z- is represented by the following formula (5):

[0127]

[Chemical 27]

(In the formula, X is S, O, NH or NR.
¹ , R ¹ represents an alkyl group having 1 to 6 carbon atoms, R is a substituent on the heterocycle, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a fluorine atom, a chlorine atom, a bromine atom, an amino group. Represents a group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. n represents an integer of 0 to 3, and when n is 2 or more,
R may be the same or different. ), And the obtained compound is the compound of the formula (2) in which R _n -Z-
Is the above formula (5)

(In the formula, X is S, O, NH or NR.
¹ , R ¹ represents an alkyl group having 1 to 6 carbon atoms, R is a substituent on the heterocycle, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a fluorine atom, a chlorine atom, a bromine atom, an amino group. Represents a group, a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. n represents an integer of 0 to 3, and when n is 2 or more,
R may be the same or different. ) Is a compound represented by.

Specific examples of such compounds include 2-furanacrylic acid, 3-furanacrylic acid, 2-thienylacrylic acid, 3-thienylacrylic acid, 2-pyrrolylacrylic acid, 3-pyrrolylacrylic acid and the like. Is mentioned.

For example, when an enzyme derived from Lithospermum erythrorhizon is used, 2-furan acrylic acid, 3
Furan acrylic acid, 2-thienyl acrylic acid, 3-thienyl acrylic acid, 2-pyrrolyl acrylic acid, 3-pyrrolyl acrylic acid are preferred, correspondingly α-amino-2
-Furanpropionic acid, α-amino-3-furanpropionic acid, α-amino-2-thienylpropionic acid, α-
Amino-3-thienylpropionic acid, α-amino-2-
Pyrrole propionic acid and α-amino-3-pyrrole propionic acid are obtained.

According to the method of the present invention, an L-amino acid having high optical purity can be efficiently obtained by a one-step reaction using an acrylic acid derivative which can be easily obtained by organic synthesis as a substrate. The L-amino acid thus obtained is useful as a synthetic intermediate in the field of fine chemicals such as pharmaceuticals and agricultural chemicals that require high optical purity.

[0133]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the examples and comparative examples, 0.
In the presence of 1 M sodium borate buffer pH 8.5 and 10 mM L-phenylalanine as a substrate, the amount of enzyme that releases 1 μmole of cinnamic acid per minute at 30 ° C. is 1 un.
It was defined as it.

The amount of enzyme protein was quantified by the Biuret method with bovine serum albumin as a standard,
The enzyme specific activity was expressed as unit / mg protein. In addition, the identification of acrylic acid derivatives and amino acids
¹ H-NMR, ¹³ C-NMR, and the reaction solution by HPLC
It was carried out by comparing the UV absorption intensity with the standard sample.

Separation and quantification of the obtained acrylic acid derivative was carried out.
The analysis was performed under the following analytical conditions. Reversed-phase HPLC analysis conditions Column: Shodex (registered trademark of Showa Denko KK)
RSpak NN-614 (manufactured by Showa Denko KK) Column temperature: 40 ° C. Eluent: acetonitrile / water / 50 mM H ₃ PO ₄ -K
H ₂ PO ₄ aqueous solution (pH 3) = 20/70/10 Flow rate: 1.0 ml / min Detection: UV absorption by 210 nm

The optical purity of the obtained amino acid was analyzed by optical resolution HPLC under the following conditions. Optical resolution HPLC analysis conditions Column: Shodex (registered trademark of Showa Denko KK)
ORpak CRX-853 (manufactured by Showa Denko KK) Column temperature: room temperature (22 ° C) Eluent: acetonitrile / water = 15/85 CuSO
₄ 2.5 mM flow rate: 1.0 ml / min. Detection method: By UV 256 nm absorption

Example 1 Reaction with Cultured Cells (Parsley) The composition of medium A is shown in Table 1.

[Table 1]

10 L of the medium A having the above composition was dispensed into an Erlenmeyer flask, and each was preliminarily subcultured in the same medium at 27 ° C. in the dark under aeration (passage 5 generations) for parsley ( Petros ).
elinum hortense ) cell culture suspension was inoculated and cultured at 27 ° C. in the dark under aeration at 200 rpm for 10 days with shaking. Further, the culture was continued for 24 hours while irradiating the light of a fluorescent lamp, and the obtained culture solution was suction-filtered with a glass fiber filter paper,
Cultured cells containing about 1.6 kg of phenylalanine ammonia lyase were obtained. The activity per fresh cell weight was 0.0051 u / g.

2 g of the obtained cultured cells was adjusted to pH by combining 2M ammonia / ammonium carbonate aqueous solution (pH = 10) containing 0.1% of various acrylic acid derivatives (2M ammonia water and 2M ammonium carbonate aqueous solution). The product was suspended in 10 mL and reacted at 30 ° C. for 24 hours with stirring. Table 2 shows products and concentrations obtained by the respective reactions, and optical purities.

Comparative Example 1: Reaction with Microorganism-Derived Enzyme In place of the cultured cells of Example 1, commercially available Rhodotorula gl
utinis- derived phenylalanine ammonia lyase (manufactured by Sigma) specific activity 0.44 unit / mg 0.025m
Table 2 shows the products and the concentrations and the optical purities when the reaction was carried out under the same conditions by adding g equivalent.

[0141]

[Table 2]

Example 2: Reaction by treated cells of cultured cells
Ceri) Parsley cultured cells obtained by culturing in the same manner as in Example 1.
5 kg was suspended in 3 L of 0.1 M sodium borate buffer (pH = 8.8) and subjected to ultrasonic crushing treatment for 20 minutes under ice cooling. Insoluble matter was removed from the treated product by 10,000
It was sedimented by centrifugation at 0 rpm for 15 minutes, and the supernatant was collected.

Crushed ammonium sulfate was gradually added to the obtained extract with stirring under ice-cooling over 30 minutes until 40% saturation, and the solution was 10,000 rp.
The precipitate was removed by centrifugation for 15 minutes. Similarly, ammonium sulfate was added to the obtained supernatant until it became 55% saturated, and the solution was centrifuged at 10,000 rpm for 15 minutes to recover the precipitate. Precipitate 150 ml of 0.05M
It was dissolved in Tris-HCl buffer and dialyzed in 100 volumes of the same buffer at 4 ° C. for 24 hours. 170m solution after dialysis
1 was obtained as a crude enzyme extract of phenylalanine ammonia lyase. The total protein amount was 620 mg and the specific activity was 0.0205 U / mg.

1 ml of the obtained crude extract solution, 9 ml of 2M ammonia / ammonium carbonate aqueous solution (pH = 10) (pH adjusted by combining 2M ammonia water and 2M ammonium carbonate aqueous solution), and various cinnamic acids were added. The derivative was added to 0.5%, and the reaction was carried out by stirring at 30 ° C. for 24 hours. Table 3 shows products and concentrations obtained by the respective reactions, and optical purities.

Comparative Example 2: Reaction with Microorganism-Derived Enzyme The commercially available Rhodotorul was used instead of the crude enzyme extract of Example 2.
a graminis- derived phenylalanine ammonia lyase (manufactured by Sigma) 0.44 units / mg 0.1
Table 3 shows the product and concentration, and the optical purity when 75 mg was added and the reaction was carried out under the same conditions.

[0146]

[Table 3]

Example 3: Reaction with treated plant tissue (Cu
cumis sativus L; cucumber) Cucumber ( Cucumis sativus L.) seeds were cultivated and germinated on a petri dish covered with filter paper moistened with water at 25 ° C. for 72 hours. Approximately 50g after 4 hours of fluorescent light irradiation
The hypocotyl was harvested.

The obtained hypocotyl tissue was ground in a mortar under ice cooling, and the ground product was further washed with 0.1 M sodium borate buffer (pH = 1 mM) containing 1 mM glutathione.
8.8) After suspending in 100 ml, ultrasonic crushing treatment was performed for 20 minutes. Insoluble matter from treated product is 10,000r
It was sedimented by centrifugation at pm for 15 minutes and the supernatant was collected.

Crushed ammonium sulfate was gradually added to the obtained extract with stirring under ice-cooling over 30 minutes until 30% saturation, and the solution was added at 10,000 rp.
The precipitate was removed by centrifugation for 15 minutes. Similarly, ammonium sulfate was added to the obtained supernatant until it reached 70% saturation, and the solution was centrifuged at 10,000 rpm for 15 minutes to recover the precipitate. Precipitate 20 ml of 0.05M T
It was dissolved in ris-HCl buffer and dialyzed in 100 volumes of the same buffer at 4 ° C. for 24 hours. 2.2 ml of solution after dialysis
Was obtained as a crude enzyme extract. Total protein amount 50.7m
The specific activity was 0.0307 U / mg.

0.1 ml of the obtained crude extract, 2M ammonia / ammonium carbonate aqueous solution (pH = 10) (2M
Ammonia water and 2M ammonium carbonate aqueous solution were combined to adjust the pH) 9.9 ml of a mixed solution was added with various cinnamic acid derivatives so as to be 0.5%, and at 30 ° C.,
The reaction was carried out by stirring for 24 hours. Table 4 shows products and concentrations obtained by each reaction, and optical purity.

Comparative Example 3: Reaction with Microorganism-Derived Enzyme As a comparative example, a commercially available Rhodotor was used instead of the crude enzyme extract.
phenylalanine ammonia lyase from ula graminis (Sigma) specific activity 0.44 unit / mg 0.1
Table 4 shows the products and the concentrations and the optical purities when 6 mg was added and the reaction was carried out under the same conditions.

[0152]

[Table 4]

Example 4; Cultivation and transformation of transformants
The transformant used in the reaction was L broth (polypeptone 1).
%, NaCl 0.5%, yeast extract 0.5%, pH 7.
0%) was added to 2% agar and plated on an agar plate medium plated overnight, and one platinum loop of the cells was cultured in 5 to 100 ml of L broth at 30 ° C. for 16 hours. After culturing the cells, the cells are washed with an equal volume of physiological saline to the culture solution, and then the cells are washed with a half volume of the reaction solution (4M ammonia / ammonium carbonate (pH 10.3)). ),
Add substrate with final concentration of 0.2% (2000mg / L) 3
The reaction was carried out at 0 ° C with shaking. A portion of this reaction solution is taken at regular intervals, the bacterial cells are removed by centrifugation, and the supernatant is
The amount of product was quantified by LC analysis.

Example 5: Preparation of cDNA library and pa
l gene acquisition Cell culture of purple ( Lithospermum erythrorhizon ),
Creation of cDNA library and acquisition of pal gene are Ya
Report by zaki et al. ( Plant Cell Physiol . (1995), 36, 1319-
1329; Biosci. Biotech . Biochem . (1997), 61 (12), 1
995-2003), a cDNA library of tea ( Camellia sinensis) was constructed and a pal gene was obtained by Matsumoto et al. ( Theor . Appl . Genet . (1994), 89 (6), 671-675).
Are described in detail in. The inventor refers to these existing information, makes some changes, and makes pa from each plant.
1 gene was obtained. The series of methods can be applied to all plant-derived pal whose nucleotide sequence is clear. As a material for obtaining mRNA, in addition to cultured cells, plant tissues under pal gene expression conditions can be used.

(Preparation of Murasaki cDNA Library) Cells prepared from Murasaki tissue were treated with LS medium (normal growth medium; indole acetic acid 10 μM and kinetin 0.1).
(including μM), the cells were cultured at 25 ° C. for 1 week. The obtained cultured cells were then subcultured in M9 medium (shikonin production medium; containing indoleacetic acid 10 μM and kinetin 0.1 μM), and further cultured for 2 days. From the obtained cultured cells, QuickPrep Micro mRNA Pu
mRNA was prepared using a Ricification Kit (manufactured by Amersham Pharmacia Biotech) according to the method of using the Kit standard. Next, RT-PCR using the obtained mRNA as a template was performed for the First-Strand cD.
NA Synthesis Kit (Amersham Pharmacia Biotech) and Oligo (dT) 12-18 primer were used. The mixture of generated DNA fragments was used as a cDNA library for gene amplification PCR template.

(Preparation of Tea cDNA Library) From the frozen young leaves of tea (collected in the sunny afternoon), DNA fragments were obtained by RT-PCR in the same manner as the above purple.
A mixture of DNA fragments is used as a cDNA for gene amplification PCR template.
A library.

(Acquisition of pal gene) Two kinds of pal genes present in purple (hereinafter referred to as "LePAL
1 "and" LePAL2 ". ) Is already Yazaki
As SEQ ID NOs: D83075 and D83076, respectively,
The pal gene present in tea (hereinafter referred to as “CAMPAL”) has been published by Matsumoto et al. As SEQ ID NO: D26596 in databases such as GenBank and EMBL. A pal gene-specific primer was prepared using the sequences at the 5'end and the 3'end. At that time, the obtained DN
The primers were designed to have appropriate restriction enzyme sites at both ends so that the A fragment could be connected to the plasmid vector.

From the restriction enzymes frequently used for the multiple cloning sites of various plasmid vectors, an appropriate one (no cutting site in the pal gene sequence) was searched, and as a result, LePAL2 was found to have an EcoRI site at the 5'end, S on 3'end
As for the alI site, CAMPAL decided to use the SmaI site at the 5'end side and the HindIII site at the 3'end side. Since there was no suitable site on the 5'end side of LePAL1, the 5'end was EcoRV, and the blunt end generated after cleavage was designed to be ligated to the blunt end of the SmaI site of the vector.
The 3'-terminal side was SalI as in LePAL2.

Primer sequences: (LePAL1)

[Sequence 1] 5'side (with EcoRV site); 23-mer oligonucleotide having the sequence shown in Sequence Listing 1

[Sequence 2] 3'side (with SalI site); 23-mer oligonucleotide having the sequence shown in Sequence Listing 2

(LePAL2)

[Sequence 3] 5'side (with EcoRI site); 25-mer oligonucleotide having the sequence shown in Sequence Listing 3

[Sequence 4] 3'side (with SalI site); 23-mer oligonucleotide having the sequence shown in Sequence Listing 4

(CAMPAL)

[Sequence 5] 5'side (with SmaI site); 23-mer oligonucleotide having the sequence shown in Sequence Listing 5

[Sequence 6] 3'side (with HindIII site); 23-mer oligonucleotide having the sequence shown in Sequence Listing 6

[0162]     Reaction solution composition:       Template cDNA 0.5-2 μg       100 pmol each       dNTP solution 1 mM each       10x reaction buffer 10 μl       ExTaq DNA Polymerase (Takara Shuzo) 2.5U Total 50 μl

Reaction conditions: Heat denaturation 94 ℃, 30 seconds Annealing 55 ℃, 60 seconds Extension 72 ° C, 120 seconds 24 cycles

When the gene fragment amplification was attempted by varying the amount of the template cDNA under the above conditions, theoretically calculated LePAL1, LePAL2, and CAMP were obtained.
A fragment with an AL fragment length of about 2.1 kb was amplified almost specifically. Each fragment was subjected to agarose gel electrophoresis, extracted, recovered, purified, and then analyzed for the nucleotide sequence using the primers used for amplification.
It was confirmed that the sequence information was consistent with AL1, LePAL2, and CAMPAL.

Example 6 of transformants showing PAL activity
Preparation The obtained gene fragment was subjected to agarose gel electrophoresis, extracted and collected. Obtained LePA with restriction enzyme cleavage site
The L1, LePAL2, and CAMPAL fragments were cleaved with restriction enzymes EcoRV and SalI, EcoRI and SalI, and SmaI and HindIII, respectively, and then subjected to agarose gel electrophoresis again to extract and recover. These fragments were designated EcoRVSalI and EcoRI-Sa, respectively.
After cutting with Il and SmaI-HindIII, it was subjected to agarose gel electrophoresis and ligated with the extracted and recovered pUC18 (FIGS. 1 and 2).

The transformants obtained by transforming Escherichia coli JM109 strain with these plasmids were transformed with isopropyl-β.
-D-thiogalactopyranoside (IPTG) 0.1 mM
And L-agar plate medium containing 0.1% X Gal,
The cells were cultured at 37 ° C for 24 hours. Several white colonies were selected from the generated colonies, and their plasmids were adjusted to confirm the restriction enzyme digestion patterns. As a result, the target plasmids pULePAL1, pULePAL2 and pU were identified.
A plurality of transformants having CAMPAL were obtained.

Three strains each were arbitrarily selected from the obtained transformants, and 5 ml of L broth was added according to the method described in Example 4.
Each strain was cultured in duplicate (2 species x 3 strains x 2 replicates). 12 hours after the start of the culture, isopropyl-β-D was added to one of the duplicates.
-Thiogalactopyranoside (IPTG) was added to the culture solution to a concentration of 0.1 mM, and the cells were further cultured for 4 hours. The obtained culture broth was centrifuged to collect the cells, and the obtained cells were subjected to the reaction by the method described in Example 4. The product after 10-hour reaction was quantified by HPLC. As a result, as shown in Table 5, each transformant was found to have phenylalanine ammonia lyase regardless of the presence of isopropyl-β-D-thiogalactopyranoside (IPTG). It showed activity (unit mg / L).

[0168]

[Table 5]

Example 7: Phenyla using a transformant
Production of Lanine Derivative Each of the pULePAL1, pULePAL2 and pUCAMPAL transformants obtained in Example 6 was cultured in 100 ml of L broth containing 100 ppm of ampicillin according to the method described in Example 4. This culture was further inoculated into a 5 L jar fermenter containing 2 L of L broth containing 100 ppm of ampicillin, 30 ° C., 800 rpm,
Aeration 1 ml / min. It was aerated and agitated for 10 to 12 hours.

The cells obtained by centrifuging the culture solution from the late logarithmic growth phase to the early stationary phase were resuspended in 1 L of 4M ammonia / ammonium carbonate (pH 10.3), and 20 g of the substrate (see Table 6) was added. In addition, the reaction was carried out with stirring at 30 ° C. and 800 rpm. A part of the reaction solution was taken every hour, and the product (see Table 6) generated in the reaction solution was quantified by HPLC. When the reaction was continued while successively adding the substrate so that the substrate concentration was kept at about 2%, 1 to 3% was accumulated in the reaction solution in about 10 hours (Table 6).

After completion of the reaction, the reaction solution was concentrated under reduced pressure with a rotary evaporator to remove excess ammonia,
Concentrated hydrochloric acid was added to adjust the pH to 1, the residual substrate was precipitated, the precipitate was filtered, and the filtrate was concentrated with a rotary evaporator. The generated precipitate was filtered with filter paper, washed with dilute hydrochloric acid (0.1N), and then dried in vacuum. The purity of this dried preparation was 99% or more. The impurities detected were cinnamic acid derivatives and acrylic acid derivatives, which are substrates in both systems. When the optical purities of these derivatives were assayed according to the method described above, only the L-form was detected and the D-form was below the detection limit.

[0172]

[Table 6]

In this Example, a plurality of phenylalanine ammonia lyases having different amino acid sequences were used (cucumber sequence is unidentified) in order to show that phenylalanine ammonia lyase of plant origin is generally effective for the present invention. Sequence homology is shown in FIG. 3, and although these are enzymes that differ from each other in their amino acid sequences by 10 to 20%, they all have sufficiently effective characteristics in the present invention, that is, they are derived from microorganisms. It was clarified that it has a much higher phenylalanine derivative conversion production ability than the enzyme.

[0174]

INDUSTRIAL APPLICABILITY According to the present invention, by using a phenylalanine ammonia-lyase derived from a plant, a corresponding amino acid can be easily and efficiently obtained from an acrylic acid derivative as a raw material, and particularly various phenyl groups on phenyl groups. The L-phenylalanine derivative having the substituent can be simply and efficiently obtained.

The L-amino acid obtained by the method for producing an L-amino acid of the present invention is useful in a wide range of fields as a chiral building block for physiologically active organic compounds such as pharmaceutical intermediates and agrochemical intermediates. It is useful as a body.

[0176]

[Brief description of drawings]

FIG. 1 is a construction diagram of a purple PAL expression plasmid showing LePAL1 and LePAL2.

FIG. 2 is a construction diagram of a ChaPAL expression plasmid showing CAMPAL.

FIG. 3 is a diagram showing an example of homology of PAL amino acid sequences.

[0177]

[Sequence list]

SEQUENCE LISTING <110> SHOWA DENKO KK <120> Biologycal Production of Phenylalanine Deriv
atives <130> Primers for PAL <140><141><160> 6 <170> PatentIn Ver. 2.1 <210> 1 <211> 23 <212> DNA <213> Lithospermum erythrorhizon <400> 1 aagatatcat ggaaaccata gtg 23 <210> 2 <211> 23 <212> DNA <213> Lithospermum erythrorhizon <400> 2 ttgtcgactt aacagattgg aag 23 <210> 3 <211> 25 <212> DNA <213> Lithospermum erythrorhizon <400> 3 aagaattcat ggaaaatgga aatgg 25 <210> 4 <211> 23 <212> DNA <213> Lithospermum erythrorhizon <400> 4 ttgtcgacta acatattgga aga 23 <210> 5 <211> 23 <212> DNA <213> Camellia sinensis <400> 5 aacccgggat ggatagtacc acc 23 <210> 6 <211> 23 <212> DNA <213> Camellia sinensis <400> 6 ttaagcttct aacagatagg aag 23

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4B024 AA03 BA07 BA71 CA04 DA06                       EA04 GA11 HA12                 4B064 AE03 AE29 AE43 AE45 AE48                       CA21 CB30 CD07 CD12 DA01                       DA11

Claims (30)

[Claims] 1. The following formula (1): (In the formula, Z represents an aromatic ring group which may contain a hetero atom, R represents a substituent on the aromatic ring, n represents an integer of 0 or more, and when n is 2 or more, R represents The phenylalanine ammonia lyase derived from a plant is allowed to act on the acrylic acid derivative represented by the same or different) in the presence of ammonia, and the following formula (2): (However, in the formula, Z represents an aromatic ring group which may contain a hetero atom, R represents a substituent on the aromatic ring, n represents an integer of 0 or more, and when n is 2 or more, R represents May be the same or different from each other). 2. R _n -Z- is represented by the following formula (3): (However, R is a substituent on the benzene ring, and a cyano group, a hydroxyl group, a carboxyl group, an amide group, a fluorine atom,
Chlorine atom, bromine atom, amino group, nitro group, hydroxymethyl group, alkyl group having 1 to 6 carbon atoms or 1 to carbon atoms
6 represents an alkoxy group. n represents an integer of 0 to 5,
When is 2 or more, R may be the same or different. The method for producing an L-amino acid according to claim 1, wherein 3. R _n -Z- is represented by the following formula (4): (However, in the formula, X represents S, O, NH or NR ¹ , and R ¹
Represents an alkyl group having 1 to 6 carbon atoms, R is a substituent on the heterocycle, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a fluorine atom, a chlorine atom, a bromine atom, an amino group,
It represents a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. n is 0
Represents an integer of 3 and when n is 2 or more, R may be the same or different. ) Is L- according to claim 1.
Amino acid manufacturing method. 4. R _n -Z- is represented by the following formula (5): (However, in the formula, X represents S, O, NH or NR ¹ , and R ¹
Represents an alkyl group having 1 to 6 carbon atoms, R is a substituent on the heterocycle, and is a cyano group, a hydroxyl group, a carboxyl group, an amide group, a fluorine atom, a chlorine atom, a bromine atom, an amino group,
It represents a nitro group, a hydroxymethyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. n is 0
Represents an integer of 3 and when n is 2 or more, R may be the same or different. ) Is L- according to claim 1.
Amino acid manufacturing method. 5. The L according to any one of claims 1 to 4, wherein a plant cultured cell containing phenylalanine ammonia lyase and / or a treatment composition thereof is used.
-Amino acid manufacturing method. 6. The method for producing an L-amino acid according to claim 1, wherein a composition for treating plant tissue containing phenylalanine ammonia lyase is used. 7. The plant-derived phenylalanine ammonia-lyase gene is allowed to exist in a plant so that it can be expressed, and a transformed plant cultivated product or a treated product of the cultivated product is used. The method for producing an L-amino acid according to 1. 8. A plant-derived phenylalanine ammonia-lyase gene is allowed to be expressed in a microorganism,
The method for producing an L-amino acid according to any one of claims 1 to 4, wherein an enzyme obtained from the transformed microorganism culture, treated product or culture is used. 9. A plant-derived phenylalanine ammonia-lyase gene is derived from Lithospermum erythrorhizon p
an amino acid sequence derived from the base sequence of the al2 gene and 7
The method for producing an L-amino acid according to claim 7 or 8, which is a gene encoding an amino acid sequence having a sequence homology of 0% or more. 10. The plant-derived phenylalanine ammonia-lyase gene is a gene encoding an amino acid sequence having 80% or more sequence homology with the amino acid sequence derived from the base sequence of the pal2 gene derived from Lithospermum erythrorhizon. 8. The method for producing an L-amino acid according to item 8. 11. The plant from which the phenylalanine ammonia-lyase gene is derived is Lithospermum and / or Camell.
The method for producing an L-amino acid according to any one of claims 1 to 8, which belongs to the genus ia . 12. The method for producing an L-amino acid according to claim 8, wherein the microorganism is a bacterium, a yeast and / or a filamentous fungus. 13. The method for producing an L-amino acid according to claim 12, wherein the bacterium is Escherichia coli. 14. The method for producing an L-amino acid according to claim 2, wherein at least one R in the formula (3) is a hydroxyl group. 15. The method for producing an L-amino acid according to claim 2, wherein at least one of the substituents R in the formula (3) is a cyano group. 16. The method for producing an L-amino acid according to claim 2, wherein at least one of the substituents R in the formula (3) is a carboxyl group. 17. The method for producing an L-amino acid according to claim 2, wherein at least one of the substituents R in the formula (3) is an amide group. 18. The method for producing an L-amino acid according to claim 2, wherein at least one of the substituents R in the formula (3) is a halogen group. 19. The method for producing an L-amino acid according to claim 2, wherein at least one of the substituents R in the formula (3) is an amino group. 20. The method for producing an L-amino acid according to claim 2, wherein at least one of the substituents R in the formula (3) is a nitro group. 21. At least one of the substituents R in the formula (3) is a hydroxymethyl group.
The method for producing an L-amino acid according to any one of 1. 22. The method for producing an L-amino acid according to claim 2, wherein at least one of the substituents R in the formula (3) is an alkyl group having 1 to 6 carbon atoms. 23. The L according to claim 2, wherein the substituent R in the formula (3) is a cyano group, a hydroxyl group, a nitro group or a carboxyl group, and n is 1. -Amino acid manufacturing method. 24. The method for producing an L-amino acid according to claim 2, wherein the L-amino acid is L-phenylalanine. 25. The method according to claim 3, wherein X in the formula (4) is O.
The method for producing an L-amino acid according to any one of 5 to 13. 26. The method according to claim 3, wherein X in the formula (4) is S.
The method for producing an L-amino acid according to any one of 5 to 13. 27. The method for producing an L-amino acid according to claim 3, wherein X in the formula (4) is NH. 28. The method for producing an L-amino acid according to claim 4, wherein X in the formula (5) is O. 29. The method for producing an L-amino acid according to claim 4, wherein X in the formula (5) is S. 30. X in the formula (5) is NH.
14. The method for producing an L-amino acid according to any one of 13 to 13.