JP2013128460A - Method for producing terephthalic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel method for synthesizing terephthalic acid.SOLUTION: There is provided a method for synthesizing terephthalic acid comprising a step of converting phenylalanine to phenylacetamide, a step of converting phenylacetamide to phenylacetic acid, a step of converting phenylacetic acid to toluene, a step of converting toluene to p-toluic acid and a step of converting p-toluic acid to terephthalic acid. Also, there is provided a method for synthesizing terephthalic acid comprising a step of converting phenylalanine to trans-cinnamic acid, a step of converting trans-cinnamic acid to benzoic acid, and a step of converting benzoic acid to terephthalic acid. Further, there is provided a method for producing terephthalic acid comprising a step of converting phenylpyruvic acid to mandelic acid, a step of converting mandelic acid to benzoic acid and a step of converting benzoic acid to terephthalic acid.

Description

  The present invention relates to a method for producing terephthalic acid. The present invention also relates to a method for producing p-toluic acid.

In general, terephthalic acid is produced by oxidizing petroleum secondary products para-xylene and acetic acid, manganese salt, cobalt salt, etc. as catalysts. However, there are concerns about oil depletion, price increases, and consumption due to environmental problems. Research on methods for producing terephthalic acid derived from biomass has been conducted due to the interest of consumers.
For example, Draths of the United States of America produces muconic acid from plant-derived biomass, such as glucose, by genetically engineering Escherichia coli, and chemically condenses it with ethylene produced by the bio-process using the Diels-Alder method. A method for producing terephthalic acid by synthesizing 1,4-dicarboxylic acid and then dehydrogenating the benzene ring is studied (Patent Document 1).
In addition, Gevo is investigating the production of biomass-derived paraxylene by chemically converting biomass-derived isobutanol (Patent Document 2). Paraxylene can be converted to terephthalic acid by existing methods.
In addition, the University of Massachusetts and Anellotech have shown that aromatic compounds containing paraxylene can be produced from biomass by heating the biomass in an oxygen-free state, and from this, biomass-derived terephthalic acid can be produced using existing methods. A manufacturing method has been developed (Patent Document 3).
Furthermore, it is also known that limonene, which is a natural terpene, can be chemically converted to p-cymene and terephthalic acid can be synthesized by a two-step oxidation reaction (Patent Document 4).

International Publication No. 2010/148081 US20110087000 US20090227823 US2013018461

  An object of the present invention is to provide a novel method for synthesizing terephthalic acid.

In order to select a synthesis route of a novel compound from a starting compound to a target compound, the present inventors create a conversion unit that converts the chemical structure of the compound into a numerical value, and a pair of the compound, and the pair of the compound A calculation unit that calculates a difference value by subtracting the numerical value for the other compound from the numerical value for one compound of the compound, the difference value of the compound pair for which a reaction related to the compound pair is known, A determination unit that compares the difference value of the compound pair for which the reaction related to the compound pair is unknown, and determines an unknown reaction of the compound pair that matches the difference value as a virtual reaction; a node indicating the compound; A preparation unit that creates a synthetic route of the compound by edges indicating the known reaction and the virtual reaction, and a combination from the starting compound to the target compound; Route, heading and selecting section for selecting the synthetic route of the compounds created, a synthetic route generation device comprising a and filed a patent application (Japanese Patent Application No. 2010-28
1794 and Japanese Patent Application No. 2011-179573).

  And this time, when this synthetic pathway creation apparatus was used and the synthetic pathway of terephthalic acid was searched, three synthetic pathways were newly discovered and the present invention was completed.

The present invention provides the following.
[1] A method for producing terephthalic acid, comprising: converting phenylalanine into trans-cinnamic acid; converting trans-cinnamic acid into benzoic acid; and converting benzoic acid into terephthalic acid.
[2] Carbon sources of terephthalic acid-producing microorganisms that have the activity of an enzyme that converts phenylalanine into trans-cinnamic acid, an enzyme that converts trans-cinnamic acid into benzoic acid, and an enzyme that converts benzoic acid into terephthalic acid A method for producing terephthalic acid, comprising culturing in a medium containing terephthalic acid, accumulating terephthalic acid in the medium or bacterial cells, and collecting terephthalic acid from the medium or bacterial cells.
[3] A method for producing terephthalic acid, comprising a step of converting phenylpyruvic acid into mandelic acid, a step of converting mandelic acid into benzoic acid, and a step of converting benzoic acid into terephthalic acid. [4] A carbon source of an enzyme capable of producing terephthalic acid and having the activity of an enzyme that converts phenylpyruvic acid to mandelic acid, an enzyme that converts mandelic acid to benzoic acid, and an enzyme that converts benzoic acid to terephthalic acid A method for producing terephthalic acid, comprising culturing in a medium containing terephthalic acid, accumulating terephthalic acid in the medium or bacterial cells, and collecting terephthalic acid from the medium or bacterial cells.
[5] Converting phenylalanine to phenylacetamide, converting phenylacetamide to phenylacetic acid, converting phenylacetic acid to toluene, converting toluene to p-toluic acid, and converting p-toluic acid to terephthalic acid A method for producing terephthalic acid, comprising the step of converting to
[6] An enzyme capable of producing p-toluic acid, converting phenylalanine to phenylacetamide, an enzyme converting phenylacetamide to phenylacetic acid, an enzyme converting phenylacetic acid to toluene, and converting toluene to p-toluic acid A microorganism having an enzyme activity is cultured in a medium containing a carbon source to produce p-toluic acid, and the obtained p-toluic acid is converted to terephthalic acid, Method.
[7] Converting phenylalanine to phenylacetamide, converting phenylacetamide to phenylacetic acid, converting phenylacetic acid to toluene, and converting toluene to p-toluic acid, Production method.
[8] An enzyme capable of producing p-toluic acid, converting phenylalanine to phenylacetamide, an enzyme converting phenylacetamide to phenylacetic acid, an enzyme converting phenylacetic acid to toluene, and converting toluene to p-toluic acid A method for producing p-toluic acid, comprising culturing a microorganism having an enzyme activity in a medium containing a carbon source to produce p-toluic acid.

The production method of terephthalic acid derived from biomass that has been known so far involves the production of intermediate products such as para-xylene by bio-methods, which are later converted to terephthalic acid by chemical synthesis methods, making the production process complicated. Therefore, there is a concern that the input energy and plant construction cost will increase.
According to the present invention, terephthalic acid or p-toluic acid can be produced directly from biomass by a bioprocess, and production cost can be expected to be reduced by simplifying the production process. In addition, it can be said that there is a significant advantage over the conventional method because the input energy related to the process can be reduced. The terephthalic acid obtained by the method of the present invention can be suitably used for the production of polyesters such as PET.

Configuration diagram of synthetic route creation device 1 The figure which shows the example at the time of grouping atom types about the compound which has carbon (C) registered in the database of KEGG The figure which shows the example at the time of grouping atom types about the compound registered in the database of KEGG The figure which shows the example at the time of counting the number of the partial structure contained in the chemical structural formula of a compound (3-Hydroxypropionate), and the number of the bonding states of a partial structure Functional configuration diagram of composite route creation device 1 Diagram showing the flow of creation of compound synthesis pathway by virtual reaction method Diagram showing an example of a graph represented by a figure Diagram showing synthesis route information Diagram showing the flow of creation of compound synthesis route by optimization method Diagram showing examples of compound synthesis routes The figure which shows the output example of the information on a synthetic route The figure which shows the synthetic pathway of the terephthalic acid discovered by this invention

  Hereinafter, the present invention will be specifically described.

<Method for producing terephthalic acid-1>
The first aspect of the method for producing terephthalic acid of the present invention includes a step of converting phenylalanine to trans-cinnamic acid (step 1a), a step of converting trans-cinnamic acid into benzoic acid (step 1b), and benzoic acid as terephthalic acid. (Step 1c).

  Step 1a is performed using an enzyme that converts phenylalanine to transcinnamic acid or a microorganism having the activity of the enzyme. The reaction is carried out using phenylalanine as a substrate, preferably at 20 to 40 ° C., and a coenzyme or the like is added as necessary.

Here, an example of an enzyme that converts phenylalanine into trans-cinnamic acid includes phenylalanine ammonia lyase. For example, Biochemistry. 2007
The activity can be measured according to the method described in Jan 30; 46 (4): 1004-12. Then, using the activity as an index, it can be purified from bacteria of the genus Anabaena, for example, by the method described in the same document. A partially purified enzyme or a fraction having the activity of the enzyme may be used. Moreover, you may use the recombinant enzyme produced by gene recombination.

  The microorganism having the enzyme activity may be a microorganism having the enzyme activity naturally, or a microorganism to which the activity of the enzyme is imparted by genetic modification or the like. The type of microorganism is not particularly limited, but Escherichia coli, Coryneform bacteria, Pseudomonas bacteria, Bacillus bacteria, Rhizobium bacteria, Lactobacillus bacteria, Succinobacillus bacteria, Anaerobiospirillum bacteria, Actinobacillus bacteria, Examples include filamentous fungi and yeast. A microorganism to which the enzyme activity is imparted acquires DNA encoding the enzyme based on a known sequence, and introduces the DNA into the microorganism by a transformation method using a vector, homologous recombination, or the like. Can be obtained. A microorganism having the activity of the enzyme can also be obtained by replacing the promoter of the gene encoding the enzyme on the chromosome of the host microorganism with a strong promoter.

Although it does not restrict | limit especially as a well-known sequence, The base sequence (sequence number 1) of GenBank accession number: FW572332.1 which codes phenylalanine ammonia lyase derived from Anabaena variabilis is illustrated. However, as long as it encodes a protein having the activity of converting phenylalanine to transcinnamic acid, it may be a homologue such as a DNA that hybridizes under stringent conditions with a DNA having a complementary sequence of the above base sequence. Further, it has 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more identity with the amino acid sequence encoded by the nucleotide sequence of GenBank accession number: FW572332.1. It may be a DNA encoding a protein having the activity of converting to cinnamic acid.

  Step 1b is performed using an enzyme that converts transcinnamic acid into benzoic acid or a microorganism having the activity of the enzyme. The reaction is carried out using transcinnamic acid as a substrate, preferably at 20 to 40 ° C., and a coenzyme or the like is added as necessary.

  Here, the enzymes that convert transcinnamic acid into benzoic acid are Enoyl-CoA hydratase (EC 4.2.1.17), 3-Hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), and 3-Ketoacyl-CoA thiolase (EC 2.3.1.16) enzyme group. In E. coli, proteins with Enoyl-CoA hydratase and 3-Hydroxyacyl-CoA dehydrogenase activities are encoded by the fadB gene, and proteins with 3-Ketoacyl-CoA thiolase activity are encoded by the fadA gene. (DiRusso CC. J Bacteriol. 1990 Nov; 172 (11): 6459-68.). About these enzymes, activity can be measured, for example according to the method of the literature, and it can refine | purify, for example from colon_bacillus | E._coli etc. using this activity as a parameter | index. A partially purified enzyme or a fraction having the activity of the enzyme may be used. Moreover, you may use the recombinant enzyme produced by gene recombination. In addition, these enzymes may be modified in substrate specificity so as to easily convert transcinnamic acid into benzoic acid, if necessary.

  The microorganism having the enzyme activity may be a microorganism having the enzyme activity naturally, or a microorganism to which the activity of the enzyme is imparted by genetic modification or the like. The type of microorganism is not particularly limited, but Escherichia coli, Coryneform bacteria, Pseudomonas bacteria, Bacillus bacteria, Rhizobium bacteria, Lactobacillus bacteria, Succinobacillus bacteria, Anaerobiospirillum bacteria, Actinobacillus bacteria, Examples include filamentous fungi and yeast. A microorganism to which the enzyme activity is imparted acquires DNA encoding the enzyme based on a known sequence, and introduces the DNA into the microorganism by a transformation method using a vector, homologous recombination, or the like. Can be obtained. A microorganism having the activity of the enzyme can also be obtained by replacing the promoter of the gene encoding the enzyme on the chromosome of the host microorganism with a strong promoter.

  Although it does not restrict | limit especially as a well-known sequence, The said fadB gene (sequence number 3) and fadA gene (sequence number 5) are illustrated. However, as long as it encodes a protein having the activity of converting transcinnamic acid to benzoic acid, it is a homologue such as DNA that hybridizes under stringent conditions with DNA having a complementary sequence of the base sequences of the fadB and fadA genes. May be. Further, it has 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more identity with the amino acid sequence encoded by the fadB and fadA genes, and transcinnamic acid to benzoic acid. It may be DNA encoding a protein having an activity to convert.

  Step 1c is performed using an enzyme that converts benzoic acid to terephthalic acid, or a microorganism having the activity of the enzyme. The reaction is carried out using benzoic acid as a substrate, preferably at 20 to 40 ° C., and a coenzyme or the like is added as necessary.

Here, the enzyme that converts benzoic acid to terephthalic acid includes an enzyme that adds a carboxyl group to the benzene ring. For this enzyme, for example, Environmental Microbio
The activity can be measured according to the method described in logy (2010) 12 (10), 2783-2796. And it can refine | purify from Closrida genus bacteria etc. by the method as described in the literature by using this activity as a parameter | index. A partially purified enzyme or a fraction having the activity of the enzyme may be used. Moreover, you may use the recombinant enzyme produced by gene recombination.

  The microorganism having the enzyme activity may be a microorganism having the enzyme activity naturally, or a microorganism to which the activity of the enzyme is imparted by genetic modification or the like. The type of microorganism is not particularly limited, but Escherichia coli, Coryneform bacteria, Pseudomonas bacteria, Bacillus bacteria, Rhizobium bacteria, Lactobacillus bacteria, Succinobacillus bacteria, Anaerobiospirillum bacteria, Actinobacillus bacteria, Examples include filamentous fungi and yeast. A microorganism to which the enzyme activity is imparted acquires DNA encoding the enzyme based on a known sequence, and introduces the DNA into the microorganism by a transformation method using a vector, homologous recombination, or the like. Can be obtained. A microorganism having the activity of the enzyme can also be obtained by replacing the promoter of the gene encoding the enzyme on the chromosome of the host microorganism with a strong promoter.

Although it does not restrict | limit especially as a well-known sequence, AbcA and AbcD gene of Table 1 are illustrated (Microbiology (2010) 12 (10), 2783-2796). It is considered that the AbcD and AbcA genes form an operon, and the encoded protein forms a hetero complex to carry out an enzymatic reaction. However, as long as it encodes a protein having an activity of converting toluene into p-toluic acid, it may be a homologue such as a DNA that hybridizes under stringent conditions with a DNA having a complementary sequence of the above base sequence. Further, it has 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more identity with the amino acid sequence encoded by the base sequence shown in Table 1, and toluene is p- It may be a DNA encoding a protein having an activity of converting to toluic acid.

  For steps 1a to 1c, each step may be performed separately using an enzyme that catalyzes each reaction, or each reaction may be performed separately using a microorganism that has the activity of the catalytic enzyme. It is preferable to carry out a conversion reaction from phenylalanine, which is a starting material, to terephthalic acid in the same reaction system using microorganisms having the activity of all enzymes that catalyze the above.

  That is, a reaction solution containing phenylalanine, which is a microorganism having an activity of converting phenylalanine into transcinnamic acid, an activity of converting transcinnamic acid into benzoic acid, and an activity of converting benzoic acid into terephthalic acid In addition to the above, terephthalic acid can usually be produced by reaction at 20 to 40 ° C. It is preferable to add a coenzyme or the like as necessary.

  In addition, you may use the processed material of microorganisms for microorganisms. Examples of the treated product include cells treated with acetone, toluene, and the like, freeze-dried cells, disrupted cells, cell-free extracts obtained by disrupting cells, and the like. Moreover, the microbial cell fixed to the support | carrier by the conventional method, this processed material, an enzyme, etc. can also be used.

  Also, using a microorganism having an ability to produce terephthalic acid, an enzyme activity that converts phenylalanine into trans-cinnamic acid, an enzyme activity that converts trans-cinnamic acid into benzoic acid, and an enzyme activity that converts benzoic acid into terephthalic acid, Terephthalic acid can also be produced from a carbon source such as glucose by fermentation. In this case, the medium and culture conditions used for culturing the microorganism can be appropriately set according to the type of microorganism used, but contain a carbon source, a nitrogen source, an inorganic salt, and other organic micronutrients as necessary. A normal medium can be used. The culture is performed under conditions suitable for the growth of the microorganism to be used. Usually, it is carried out at a culture temperature of 10 ° C to 45 ° C for 12 to 96 hours. “Having the ability to produce terephthalic acid” means having the ability to assimilate the carbon source and accumulate terephthalic acid in the medium or cells when cultured in a medium containing a carbon source. For example, in a known phenylalanine producing bacterium, by increasing the enzyme activity for converting phenylalanine to transcinnamic acid, the enzyme activity for converting transcinnamic acid to benzoic acid, and the enzyme activity for converting benzoic acid to terephthalic acid, Productivity can be imparted.

The carbon source is not particularly limited as long as it is a carbon source that can be assimilated by the microorganism. For example, carbohydrates such as galactose, lactose, glucose, fructose, glycerol, sucrose, saccharose, starch, cellulose; glycerin, mannitol, xylitol, Fermentable carbohydrates such as polyalcohols such as ribitol are used.
As the nitrogen source, for example, peptone, meat extract, yeast extract, corn steep liquor and the like are used in addition to ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate.
As the inorganic salt, for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride and the like are used.
In addition, vitamins such as biotin, pantothenic acid, inositol, and nicotinic acid, nucleotides, amino acids, and other factors that promote growth may be added as necessary.

  Terephthalic acid can be recovered and purified from the culture medium by methods well known in the art, such as extraction with an organic solvent, distillation, column chromatography, and the like.

<Method for producing terephthalic acid-2>
The second aspect of the method for producing terephthalic acid according to the present invention includes a step of converting phenylpyruvic acid to mandelic acid (step 2a), a step of converting mandelic acid to benzoic acid (step 2b), and benzoic acid to terephthalic acid. (Step 2c).

  Step 2a is performed using an enzyme that converts phenylpyruvic acid to mandelic acid or a microorganism having the activity of the enzyme. The reaction is carried out using phenylpyruvic acid as a substrate, preferably at 20 to 40 ° C., and a coenzyme or the like is added as necessary.

Here, as an enzyme that converts phenylpyruvic acid to mandelic acid, hydroxymandelate
synthase (HMS) (EC1.13.11.46). For this enzyme, the activity can be measured, for example, according to the method described in J. Am. Chem. Soc. 2000, 122, 5389-5390. And it can refine | purify from Amycolatopsis genus bacteria etc. by the method as described in the literature using this activity as a parameter | index. A partially purified enzyme or a fraction having the activity of the enzyme may be used. Moreover, you may use the recombinant enzyme produced by gene recombination.

  The microorganism having the enzyme activity may be a microorganism having the enzyme activity naturally, or a microorganism to which the activity of the enzyme is imparted by genetic modification or the like. The type of microorganism is not particularly limited, but Escherichia coli, Coryneform bacteria, Pseudomonas bacteria, Bacillus bacteria, Rhizobium bacteria, Lactobacillus bacteria, Succinobacillus bacteria, Anaerobiospirillum bacteria, Actinobacillus bacteria, Examples include filamentous fungi and yeast. A microorganism to which the enzyme activity is imparted acquires DNA encoding the enzyme based on a known sequence, and introduces the DNA into the microorganism by a transformation method using a vector, homologous recombination, or the like. Can be obtained. A microorganism having the activity of the enzyme can also be obtained by replacing the promoter of the gene encoding the enzyme on the chromosome of the host microorganism with a strong promoter.

  Although it does not restrict | limit especially as a well-known sequence, The base sequence (sequence number 11) of GenBank accession number AJ223998.1: 14957..16030 which codes hydroxymandelate synthase derived from Amycolatopsis orientalis is illustrated. However, as long as it encodes a protein having an activity of converting phenylpyruvic acid to mandelic acid, it may be a homologue such as DNA that hybridizes under stringent conditions with DNA having a complementary sequence of the above base sequence. Further, it has 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more identity with the amino acid sequence encoded by the nucleotide sequence of GenBank accession number AJ223998.1, and phenylpyrubin It may be a DNA encoding a protein having an activity of converting an acid into mandelic acid. If necessary, the substrate specificity may be modified using a protein engineering technique or the like so that phenylpyruvic acid can be easily converted to mandelic acid. 4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27), which has a similar steric structure to hydroxymandelate synthase, can also be used to convert phenylpyruvic acid to mandelic acid.

  Step 2b is performed using an enzyme that converts mandelic acid to benzoic acid or a microorganism having the activity of the enzyme. The reaction is performed using mandelic acid as a substrate, preferably at 20 to 40 ° C., and a coenzyme or the like is added as necessary.

Here, examples of the enzyme that converts mandelic acid into benzoic acid include an enzyme group that catalyzes the reaction of the mandelic acid pathway. That is, (S) -Mandelate dehydrogenase converts (S) -mandelic acid to benzoylformate (EC 1.1.99.31), benzoylformate decarboxylates to benzaldehyde (EC 4.1.1.7), and Benzaldehyde dehydrogenase converts benzaldehyde. To obtain benzoic acid (EC 1.2.1.28, 1.2.1.7). Genes encoding these enzymes have been found in various microbial species, and any microorganism-derived enzyme can be used. For example, in Pseudomonas putida, (S) -Mandelate dehydrogenase is encoded in the mdlB gene (SEQ ID NO: 13), Benzoylformate decarboxylase is encoded in the mdlC gene (SEQ ID NO: 15), and NAD ⁺ -dependent benzaldehyde dehydrogenase is encoded in the mdlD gene (SEQ ID NO: 17). (Tsou AY. Et al Biochemistry. 1990 Oct 23; 29 (42): 9856-62.). In Pseudomonas stutzeri, Benzoylformate decarboxylase is encoded by the dpgB gene and NAD ⁺ / NADP ⁺ -dependent benzaldehyde dehydrogenase is encoded by the dpgC gene (Saehuan C. et al Biochim Biophys Acta. 2007 Nov; 1770 (11): 1585- 92.).

  In addition, as a route for producing benzoic acid from (S) -mandelic acid, reactions of biosynthetic pathways of antibiotics chlorobiocin and novobiocin are also candidates. CloR from Streptomyces roseochromogenes is involved in the synthesis of 3-dimethylallyl-4-hydroxybenzoate (3DMA-4HB), which is the Ring A site of chlorobiocin, and decarboxylates 3-dimethylallyl-4-hydroxymandelic acid (3DMA-4HMA) Has been reported to be converted to 3DMA-4HB (Pojer F. et al J Biol Chem. 2003 Aug 15; 278 (33): 30661-8.). CloR accepts (S) -mandelic acid as a substrate, but 3DMA-4HMA differs from (S) -mandelic acid in that it has a dimethylallyl group at the 3-position and a hydroxyl group at the 4-position. it's possible. In addition, modifying the substrate specificity of CloR as necessary can be an effective means. In addition, NovRR derived from Streptomyces spheroides has 95% homology with CloR derived from Streptomyces roseochromogenes and is thought to be involved in the synthesis of 3DMA-4HB, which is the ring A site of novobiocin (Keller S. et al Acta Crystallogr D Biol Crystallogr. 2006 Dec; 62 (Pt 12): 1564-70.). NovR, like CloR, is a candidate enzyme that produces benzoic acid from (S) -mandelic acid.

  About these enzymes, activity can be measured according to the method as described in the literature mentioned above, for example. Then, it can be purified from bacteria belonging to the genus Pseudomonas by the method described in the same literature using the activity as an index. A partially purified enzyme or a fraction having the activity of the enzyme may be used. Moreover, you may use the recombinant enzyme produced by gene recombination.

  The microorganism having the enzyme activity may be a microorganism having the enzyme activity naturally, or a microorganism to which the activity of the enzyme is imparted by genetic modification or the like. The type of microorganism is not particularly limited, but Escherichia coli, Coryneform bacteria, Pseudomonas bacteria, Bacillus bacteria, Rhizobium bacteria, Lactobacillus bacteria, Succinobacillus bacteria, Anaerobiospirillum bacteria, Actinobacillus bacteria, Examples include filamentous fungi and yeast. A microorganism to which the enzyme activity is imparted acquires DNA encoding the enzyme based on a known sequence, and introduces the DNA into the microorganism by a transformation method using a vector, homologous recombination, or the like. Can be obtained. A microorganism having the activity of the enzyme can also be obtained by replacing the promoter of the gene encoding the enzyme on the chromosome of the host microorganism with a strong promoter.

  Although it does not restrict | limit especially as a well-known sequence, The base sequence which codes the above-mentioned well-known enzyme is illustrated. However, as long as it encodes a protein having the activity of converting mandelic acid to benzoic acid, it may be a homologue such as DNA that hybridizes under stringent conditions with DNA having a complementary sequence of the above base sequence. Further, it has 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more identity with the amino acid sequence encoded by the above base sequence, and converts mandelic acid to benzoic acid. It may be DNA encoding a protein having activity.

  Step 2c is performed using an enzyme that converts benzoic acid to terephthalic acid, or a microorganism having the activity of the enzyme. The reaction is carried out using benzoic acid as a substrate, preferably at 20 to 40 ° C., and a coenzyme or the like is added as necessary.

  Here, the enzyme that converts benzoic acid to terephthalic acid includes an enzyme that adds a carboxyl group to the benzene ring. The activity of this enzyme can be measured according to the method described in Environmental Microbiology (2010) 12 (10), 2783-2796, for example. And it can refine | purify from Closrida genus bacteria etc. by the method as described in the literature by using this activity as a parameter | index. A partially purified enzyme or a fraction having the activity of the enzyme may be used. Moreover, you may use the recombinant enzyme produced by gene recombination.

  The microorganism having the enzyme activity may be a microorganism having the enzyme activity naturally, or a microorganism to which the activity of the enzyme is imparted by genetic modification or the like. The type of microorganism is not particularly limited, but Escherichia coli, Coryneform bacteria, Pseudomonas bacteria, Bacillus bacteria, Rhizobium bacteria, Lactobacillus bacteria, Succinobacillus bacteria, Anaerobiospirillum bacteria, Actinobacillus bacteria, Examples include filamentous fungi and yeast. A microorganism to which the enzyme activity is imparted acquires DNA encoding the enzyme based on a known sequence, and introduces the DNA into the microorganism by a transformation method using a vector, homologous recombination, or the like. Can be obtained. A microorganism having the activity of the enzyme can also be obtained by replacing the promoter of the gene encoding the enzyme on the chromosome of the host microorganism with a strong promoter.

Although it does not restrict | limit especially as a well-known sequence, AbcA and AbcD gene of Table 2 are illustrated. It is considered that the AbcD and AbcA genes form an operon, and the encoded protein forms a hetero complex to carry out an enzymatic reaction. However, as long as it encodes a protein having an activity of converting toluene into p-toluic acid, it may be a homologue such as a DNA that hybridizes under stringent conditions with a DNA having a complementary sequence of the above base sequence. Further, it has 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more identity with the amino acid sequence encoded by the base sequence described in the table, and toluene is p-toluyl. It may be a DNA encoding a protein having an activity of converting to an acid.

  About process 2a-2c, although each reaction may be performed separately, each process may be performed separately using the enzyme which catalyzes each reaction, or each reaction using microorganisms which have the activity of a catalytic enzyme, It is preferable to carry out a conversion reaction from phenylpyruvic acid, which is a starting material, to terephthalic acid in the same reaction system using all the enzymes that catalyze each reaction or microorganisms having the activities of all the enzymes that catalyze each reaction.

  That is, a microorganism having an activity of converting phenylpyruvic acid to mandelic acid, an activity of converting mandelic acid to benzoic acid, and an activity of converting benzoic acid to terephthalic acid, or a treated product thereof, includes phenylpyruvic acid. In addition to the reaction solution, terephthalic acid can be produced usually by reacting at 20 to 40 ° C. It is preferable to add a coenzyme or the like as necessary.

  In addition, you may use the processed material of microorganisms for microorganisms. Examples of the treated product include cells treated with acetone, toluene, and the like, freeze-dried cells, disrupted cells, cell-free extracts obtained by disrupting cells, and the like. Moreover, the microbial cell fixed to the support | carrier by the conventional method, this processed material, an enzyme, etc. can also be used.

Also, using a microorganism having an ability to produce terephthalic acid, an enzyme activity that converts phenylpyruvic acid to mandelic acid, an enzyme activity that converts mandelic acid to benzoic acid, and an enzyme activity that converts benzoic acid to terephthalic acid, Terephthalic acid can also be produced from a carbon source such as glucose by fermentation. In this case, the medium and culture conditions used for culturing the microorganism can be appropriately set according to the type of microorganism used, but contain a carbon source, a nitrogen source, an inorganic salt, and other organic micronutrients as necessary. A normal medium can be used. The culture is performed under conditions suitable for the growth of the microorganism to be used. Usually, it is carried out at a culture temperature of 10 ° C to 45 ° C for 12 to 96 hours. “Having the ability to produce terephthalic acid” means having the ability to assimilate the carbon source and accumulate terephthalic acid in the medium or cells when cultured in a medium containing a carbon source. For example, in a known phenylpyruvic acid-producing bacterium, by increasing the enzyme activity for converting phenylpyruvic acid to mandelic acid, the enzyme activity for converting mandelic acid to benzoic acid, and the enzyme activity for converting benzoic acid to terephthalic acid, Can provide terephthalic acid production ability.
The medium components and terephthalic acid recovery and purification methods are as described above.

<Method for producing terephthalic acid-3>
The third aspect of the method for producing terephthalic acid of the present invention includes a step of converting phenylalanine to phenylacetamide (step 3a), a step of converting phenylacetamide to phenylacetic acid (step 3b), and a step of converting phenylacetic acid to toluene. (Step 3c), a step of converting toluene into p-toluic acid (step 3d), and a step of converting p-toluic acid into terephthalic acid (step 3e).

  Step 3a is performed using an enzyme that converts phenylalanine to phenylacetamide, or a microorganism having the activity of the enzyme. The reaction is carried out using phenylalanine as a substrate, preferably at 20 to 40 ° C., and a coenzyme or the like is added as necessary.

  L-Phenylalanine oxidase (EC1.13.12.9) is mentioned as an enzyme which converts phenylalanine to phenylacetamide. The activity of this enzyme can be measured, for example, according to the method described in Suzuki H et al (2004) J Biochem 136 (5): 617-27. Then, using the activity as an index, it can be purified from Pseudomonas bacteria by the method described in J. Biochem. (Tokyo) 92 (1982) 1235-1240. A partially purified enzyme or a fraction having the activity of the enzyme may be used. Moreover, you may use the recombinant enzyme produced by gene recombination.

  The microorganism having the enzyme activity may be a microorganism having the enzyme activity naturally, or a microorganism to which the activity of the enzyme is imparted by genetic modification or the like. The type of microorganism is not particularly limited, but Escherichia coli (Escherichia bacteria), Coryneform bacteria, Pseudomonas bacteria, Bacillus bacteria, Rhizobium bacteria, Lactobacillus bacteria, Succinobacillus bacteria, Anaerobiospirilla bacteria Examples include Actinobacillus bacteria, filamentous fungi, yeast, and the like. A microorganism to which the enzyme activity is imparted acquires DNA encoding the enzyme based on a known sequence, and introduces the DNA into the microorganism by a transformation method using a vector, homologous recombination, or the like. Can be obtained. A microorganism having the activity of the enzyme can also be obtained by replacing the promoter of the gene encoding the enzyme on the chromosome of the host microorganism with a strong promoter.

  Although it does not restrict | limit especially as a well-known sequence, The base sequence (sequence number 19) of GenBank accession number: AB167410 which codes L-Phenylalanine oxidase of Pseudomonas sp. P-501 is illustrated. However, as long as it encodes a protein having the activity of converting phenylalanine to phenylacetamide, it may be a homologue such as DNA that hybridizes under stringent conditions with DNA having a complementary sequence of the above base sequence. Here, as stringent conditions, it corresponds to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, which is a normal washing condition for Southern hybridization. The conditions for hybridizing at a salt concentration are the same (hereinafter the same). Further, it has 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more identity with the amino acid sequence encoded by the base sequence of GenBank accession number: AB167410, and phenylalanine is substituted with phenylalanine. It may be a DNA encoding a protein having an activity of converting to acetamide.

  Step 3b is performed using an enzyme that converts phenylacetamide to phenylacetic acid, or a microorganism having the activity of the enzyme. The reaction is carried out using phenylacetamide as a substrate, preferably at 20 to 40 ° C., and a coenzyme or the like is added as necessary.

  Here, an amidase (EC 3.5.1.4) is mentioned as an enzyme which converts phenylacetamide into phenylacetic acid. The activity of this enzyme can be measured according to the method described in J Bacteriol 178 (12): 3501-7 and J. Microbiol. Biotechnol. 18 (3) 552-9, for example. And it can refine | purify from Rhodococcus genus bacteria etc. by the method as described in the same literature by using this activity as a parameter | index. A partially purified enzyme or a fraction having the activity of the enzyme may be used. Moreover, you may use the recombinant enzyme produced by gene recombination.

  The microorganism having the enzyme activity may be a microorganism having the enzyme activity naturally, or a microorganism to which the activity of the enzyme is imparted by genetic modification or the like. The type of microorganism is not particularly limited, but Escherichia coli, Coryneform bacteria, Pseudomonas bacteria, Bacillus bacteria, Rhizobium bacteria, Lactobacillus bacteria, Succinobacillus bacteria, Anaerobiospirillum bacteria, Actinobacillus bacteria, Examples include filamentous fungi and yeast. A microorganism to which the enzyme activity is imparted acquires DNA encoding the enzyme based on a known sequence, and introduces the DNA into the microorganism by a transformation method using a vector, homologous recombination, or the like. Can be obtained. A microorganism having the activity of the enzyme can also be obtained by replacing the promoter of the gene encoding the enzyme on the chromosome of the host microorganism with a strong promoter.

  Although it does not restrict | limit especially as a well-known sequence, The base sequence (sequence number 21) of GenBank accession number: EU029986.1 which codes amidase derived from Rhodococcus erythropolis is illustrated. However, as long as it encodes a protein having the activity of converting phenylacetamide to phenylacetic acid, it may be a homologue such as DNA that hybridizes under stringent conditions with DNA having a complementary sequence of the above base sequence. In addition, it has 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more identity with the amino acid sequence encoded by the nucleotide sequence of GenBank accession number: EU029986.1. It may be DNA encoding a protein having an activity of converting acetamide to phenylacetic acid. Several other Rhodococcus-derived amidase genes have also been reported and can also be used to convert phenylacetamide to phenylacetic acid.

  Step 3c is performed using an enzyme that converts phenylacetic acid to toluene, or a microorganism having the activity of the enzyme. The reaction is carried out using phenylacetic acid as a substrate, preferably at 20 to 40 ° C., and a coenzyme or the like is added as necessary.

  Here, examples of the enzyme that converts phenylacetic acid into toluene include 4-hydroxyphenylacetic acid decarboxylase (EC 4.1.1.83). The activity of this enzyme can be measured according to the method described in Eur. J. Biochem 268 1363-1372 and Biochemistry 2006 45 9584-92, for example. And it can refine | purify from Clostridium genus, Tormonas genus, Lactobacillus genus bacteria, etc. by the method as described in the literature by using this activity as an index. A partially purified enzyme or a fraction having the activity of the enzyme may be used. Moreover, you may use the recombinant enzyme produced by gene recombination.

  The microorganism having the enzyme activity may be a microorganism having the enzyme activity naturally, or a microorganism to which the activity of the enzyme is imparted by genetic modification or the like. The type of microorganism is not particularly limited, but Escherichia coli, Coryneform bacteria, Pseudomonas bacteria, Bacillus bacteria, Rhizobium bacteria, Lactobacillus bacteria, Succinobacillus bacteria, Anaerobiospirillum bacteria, Actinobacillus bacteria, Examples include filamentous fungi and yeast. A microorganism to which the enzyme activity is imparted acquires DNA encoding the enzyme based on a known sequence, and introduces the DNA into the microorganism by a transformation method using a vector, homologous recombination, or the like. Can be obtained. A microorganism having the activity of the enzyme can also be obtained by replacing the promoter of the gene encoding the enzyme on the chromosome of the host microorganism with a strong promoter.

Although it does not restrict | limit especially as a well-known sequence, The base sequence of the GenBank accession number of Table 3 is illustrated. That is, it has been shown that Clodtridium difficle HpdB and HpdC proteins form a heterocomplex to form the body of decarboxylase, and that HpdA protein acts as an activator, so these genes can be used. Also, Clostridium scatologenes CsdB
, CsdC, CsdA genes can also be used. In addition, the whole genome of Tolumonas auensis, a toluene-degrading bacterium, has been reported (Lucus S et al (2009) EMBL database Accession No CP001616), and the gene group of Tolumonas auensis can also be used.
However, as long as it encodes a protein having an activity of converting phenylacetic acid into toluene, it may be a homologue such as DNA that hybridizes under stringent conditions with DNA having a complementary sequence of these base sequences. Further, it has 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more identity with the amino acid sequence (encoded by the base sequence) of the GenBank accession numbers listed in Table 3. Alternatively, it may be a DNA encoding a protein having an activity of converting phenylacetic acid into toluene.

4-Hydroxyphenylacetic acid decarboxylase (4.1.1.61) produces phenol using 4-hydroxybenzene as a substrate. It is thought that this enzyme can also convert phenylacetic acid into toluene. Sequences have been reported and are listed in Table 4.

In addition, oxaloacetate decarboxylase (EC 4.1.1.3) performs a reaction to cleave oxaloacetate into pyruvate and carbon dioxide, and at the same time has a sodium pump to convert the energy obtained at the time of cleavage into a concentration gradient. It is thought that it can be used for the reaction which converts phenylacetic acid into toluene. This enzyme requires biotin and zinc ions. This is illustrated in Table 5.

It is likely that malonate decarboxylase (EC 4.1.1.61), which has a similar sodium pump system, can carry out a reaction to convert phenylacetic acid to toluene. This is illustrated in Table 6.

  Step 3d is performed using an enzyme that converts toluene into p-toluic acid or a microorganism having activity of the enzyme. The reaction is carried out using toluene as a substrate, preferably at 20 to 40 ° C., and a coenzyme or the like is added as necessary.

  Here, the enzyme that converts toluene into p-toluic acid includes an enzyme that adds a carboxyl group to the benzene ring. The activity of this enzyme can be measured according to the method described in Environmental Microbiology (2010) 12 (10), 2783-2796, for example. And it can refine | purify from Closrida genus bacteria etc. by the method as described in the literature by using this activity as a parameter | index. A partially purified enzyme or a fraction having the activity of the enzyme may be used. Moreover, you may use the recombinant enzyme produced by gene recombination.

  The microorganism having the enzyme activity may be a microorganism having the enzyme activity naturally, or a microorganism to which the activity of the enzyme is imparted by genetic modification or the like. The type of microorganism is not particularly limited, but Escherichia coli, Coryneform bacteria, Pseudomonas bacteria, Bacillus bacteria, Rhizobium bacteria, Lactobacillus bacteria, Succinobacillus bacteria, Anaerobiospirillum bacteria, Actinobacillus bacteria, Examples include filamentous fungi and yeast. A microorganism to which the enzyme activity is imparted acquires DNA encoding the enzyme based on a known sequence, and introduces the DNA into the microorganism by a transformation method using a vector, homologous recombination, or the like. Can be obtained. A microorganism having the activity of the enzyme can also be obtained by replacing the promoter of the gene encoding the enzyme on the chromosome of the host microorganism with a strong promoter.

Although it does not restrict | limit especially as a well-known sequence, AbcA and AbcD gene of Table 7 are illustrated. It is considered that the AbcD and AbcA genes form an operon, and the encoded protein forms a hetero complex to carry out an enzymatic reaction. However, as long as it encodes a protein having an activity of converting toluene into p-toluic acid, it may be a homologue such as a DNA that hybridizes under stringent conditions with a DNA having a complementary sequence of the above base sequence. Further, it has 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more identity with the amino acid sequence encoded by the base sequence described in the table, and toluene is p-toluyl. It may be a DNA encoding a protein having an activity of converting to an acid.

  In step 3e, p-toluic acid is converted to terephthalic acid. For the conversion of p-toluic acid to terephthalic acid, for example, a method described in US Pat. No. 3,089,906 (1963) is known. A p-toluic acid ester obtained by allowing methanol or the like to coexist in the reaction system is synthesized, converted into dimethyl terephthalic acid by an oxidation reaction, and further purified to a high purity to obtain terephthalic acid.

  For steps 3a to 3d, each step may be performed separately using an enzyme that catalyzes each reaction or a microorganism having an activity of an enzyme that catalyzes each reaction, but all the enzymes that catalyze each reaction, or It is preferable to carry out a conversion reaction from phenylalanine, which is a starting material, to p-toluic acid in the same reaction system using microorganisms having the activity of all enzymes that catalyze each reaction.

  That is, an activity of converting phenylalanine to phenylacetamide, an activity of converting phenylacetamide to phenylacetic acid, an activity of converting phenylacetic acid to toluene, and a microbial cell having an activity of converting toluene to p-toluic acid or its treatment The product is added to a reaction solution containing phenylalanine, and the reaction is usually carried out at 20 to 40 ° C. to produce p-toluic acid. It is preferable to add a coenzyme or the like as necessary.

In addition, you may use the processed material of microorganisms for microorganisms. Examples of the treated product include cells treated with acetone, toluene, and the like, freeze-dried cells, disrupted cells, cell-free extracts obtained by disrupting cells, and the like. Moreover, the microbial cell fixed to the support | carrier by the conventional method, this processed material, an enzyme, etc. can also be used.
And terephthalic acid can be obtained by converting the obtained p-toluic acid into terephthalic acid.

On the other hand, it has the ability to produce p-toluic acid, has an enzymatic activity to convert phenylalanine to phenylacetamide, an enzymatic activity to convert phenylacetamide to phenylacetic acid, an enzymatic activity to convert phenylacetic acid to toluene, and toluene to p-toluic acid It is also possible to produce terephthalic acid by producing p-toluic acid from a carbon source such as glucose by a fermentation method using a microorganism having an enzyme activity to be converted into terephthalic acid, and converting the obtained p-toluic acid into terephthalic acid it can. In this case, the medium and culture conditions used for culturing the microorganism can be appropriately set according to the type of microorganism used, but contain a carbon source, a nitrogen source, an inorganic salt, and other organic micronutrients as necessary. A normal medium can be used. The culture is performed under conditions suitable for the growth of the microorganism to be used. Usually, it is carried out at a culture temperature of 10 ° C to 45 ° C for 12 to 96 hours. It is preferable that the pH is appropriately adjusted within a preferable range for microbial growth and substance production. In addition, “having the ability to produce p-toluic acid” has the ability to assimilate the carbon source and accumulate p-toluic acid in the medium or cells when cultured in a medium containing a carbon source. That means. For example, in a known phenylalanine-producing bacterium, enzyme activity for converting phenylalanine to phenylacetamide, enzyme activity for converting phenylacetamide to phenylacetic acid, enzyme activity for converting phenylacetic acid to toluene, and converting toluene to p-toluic acid By increasing the enzyme activity, p-toluic acid producing ability can be imparted.
The medium components and the method for purifying terephthalic acid are as described above. p-Toluic acid can also be recovered and purified from the culture medium by methods well known in the art, such as extraction using organic solvents, distillation, column chromatography, and the like.

Reference Example Hereinafter, a synthetic route creating apparatus used for finding a synthetic route of terephthalic acid in the present invention will be described with reference to the drawings.

<Configuration of Composite Route Creation Device 1>
FIG. 1 is a configuration diagram of the synthetic route creation apparatus 1. As shown in FIG. 1, the composite path creation device 1 has a CPU (Central Processing Unit) 2, a memory 3, a hard disk drive device 4, a portable medium drive device 5, an input device 6, a display device 7, and an external interface 8. doing.

  The CPU 2 executes the computer program stored in the memory 3 and processes the data stored in the memory 3 to control the composite path creation device 1. The memory 3 stores a program executed by the CPU 2 and data processed by the CPU 2. The memory 3 includes a volatile RAM (Random Access Memory) and a nonvolatile ROM (Read Only Memory).

The hard disk drive 4 stores a program loaded into the memory 3. Further, the hard disk drive device 4 stores data processed by the CPU 2. The portable medium driving device 5 is a driving device for a portable medium such as a CD (Compact Disc), a DVD (Digital Versatile Disk), an HD-DVD (High Definition-DVD), and a Blu-ray disc. Further, the portable medium driving device 5 may be an input / output device for a card medium having a nonvolatile memory such as a flash memory. The portable medium driven by the portable medium drive device 5 holds, for example, a computer program installed in the hard disk drive device 4 and input data. The input device 6 is, for example, a keyboard, a mouse, a pointing device, a wireless remote controller, or the like.

  The display device 7 displays data processed by the CPU 2 and data stored in the memory 3. The display device 7 is, for example, a liquid crystal display device, a plasma display panel, a CRT (Cathode Ray Tube), an electroluminescence panel, or the like.

  The external interface 8 connects the synthetic route creation device 1 to an external network. The external interface 8 includes a communication device such as a modem and a terminal adapter, for example, and controls communication between the combined route creation device 1 and devices on the external network. The external network is, for example, the Internet or a local area network (LAN). In addition, the external network may be configured by a communication line such as a telephone line, a dedicated line, an optical communication network, or a communication hygiene.

  The synthesis route creation device 1 creates a compound synthesis route from a starting compound to a target compound. There are two types of compound synthesis pathway creation: a virtual reaction method and an optimization method. In the virtual reaction method and the optimization method, a compound is defined by a unique expression method.

<Example of procedure for defining compounds by original expression method>
First, an example of a procedure for defining a compound by an original expression method will be described. In this procedure, partial structures included in the chemical structural formula of the compound are classified according to a predetermined standard, and the partial structures included in the chemical structural formula of the compound are defined as character strings. The predetermined standard may be a standard that is strictly defined for the compound structure, or may be a standard that takes into account the similarity of the compound structure. Further, the predetermined standard may be a standard based on functional group classification (alkane, alkene, etc.). For example, the partial structure included in the chemical structural formula of the compound may be classified according to the atom types registered in the database of KEGG (Kyoto Encyclopedia of Genes and Genomes). Further, for example, atom types registered in the KEGG database may be grouped, and the partial structures included in the chemical structural formulas of the compounds may be classified according to the grouped atom types.

  FIG. 2 shows an example in which atom types for compounds having carbon (C) registered in the KEGG database are grouped. In FIG. 2, atom types are grouped on the basis of chemical reaction similarity, but atom types may be grouped on the basis of functional groups (alkane, alkene, etc.). C1a, C1b, C1c, and C1d in FIG. 2 are identification numbers assigned to atom types registered in the KEGG database. The same applies to C2a and C3a in FIG. C01 in FIG. 2 is an identification number assigned to data (information) obtained by grouping C1a, C1b, C1c, and C1d. The same applies to C02 and C03 in FIG.

  FIG. 3 shows an example in which atom types for compounds registered in the KEGG database are grouped. In the field of the identification number A in FIG. 3, the identification number assigned to the atom types for the compound registered in the KEGG database is input. In the field of the identification number B in FIG. 3, an identification number assigned to data obtained by grouping atom types for compounds registered in the KEGG database is input.

  FIG. 4 shows an example of counting the number of partial structures and the number of bonded states of the partial structures included in the chemical structural formula of the compound (3-Hydroxypropionate). In FIG. 4, the number of partial structures included in the chemical structural formula of the compound (3-Hydroxypropionate) is counted for each type of partial structure, and is included in the chemical structural formula of the compound (3-Hydroxypropionate). The number of bonding states of the partial structures is counted for each type of partial structure and each type of bonding state.

In the field denoted by reference numeral 101 in FIG. 4, an identification number assigned to data obtained by grouping atom types for compounds registered in the KEGG database is input. In the field denoted by reference numeral 102 in FIG. 4, a numerical value is input after the number of partial structures included in the chemical structural formula of the compound is counted for each type of partial structure. Also, in the field denoted by reference numeral 102 in FIG. 4, a numerical value after counting the number of partial structure bonding states included in the chemical structural formula of the compound for each partial structure type and bonding state type is input. Has been. In FIG. 4, single bonds are represented by −, double bonds are represented by =, and triple bonds are represented by%. A numerical value continuously input in the field denoted by reference numeral 102 in FIG. 4 is denoted as UF (Universal Feature). As shown in FIG. 4, the UF of a compound includes atomic information indicating the number of partial structures included in the chemical structural formula of the compound and the number of bonding states of the partial structures included in the chemical structural formula of the compound. The combination information shown in FIG.

<Functional configuration of composite route creation apparatus 1>
FIG. 5 is a functional configuration diagram of the synthetic route creation apparatus 1. The synthetic route creation apparatus 1 shown in FIG. 5 includes an operation unit 10 that receives a user operation and operates the synthetic route creation device 1, a compound database 11, a known reaction database 12, a partial structure database 13, a common structure database 14, and a UF. The storage part 20 which has the database 15, the difference databases 16, 17 and the virtual reaction database 18, the control part 21 which performs various processes, and the display part 22 which displays various information are provided. Each of these functional units is realized by a computer program executed by the CPU 2, the memory 3, and the CPU 2.

<Compound Database 11>
In the compound database 11, the name of the compound and the data of the chemical structural formula of the compound are stored in association with each other. For example, the compound name registered in the KEGG database and the chemical structure data of the compound may be associated with each other and stored in the compound database 11. Further, in the compound database 11, in addition to the compounds registered in the KEGG database, the names of the compounds of the target compounds (including non-natural compounds) and the chemical structural formula data of the compounds are stored in association with each other. Good. Further, for example, the name of a compound registered in a metabolite database such as PubChem, BRENDA, or MetaCyc may be stored in the compound database 11 in association with the chemical structural formula data of the compound. Not limited to this, the names of compounds registered in various chemical substance databases and the chemical structural formula data of the compounds may be associated with each other and stored in the compound database 11.

  For a compound stored in the compound database 11, a reaction in a compound pair that is known to synthesize a compound (for example, Compound B) from a compound (for example, Compound A) is referred to as a known reaction. The user can set whether or not the reaction is a known reaction. For example, a reaction registered in the KEGG database may be a known reaction.

  For a compound stored in the compound database 11, a compound pair in which a compound (eg, compound B) is known to be synthesized from a compound (eg, compound A) is referred to as a known compound pair. At this time, compound A ′, which is an intermediate, may exist between compound A and compound B.

  For a compound stored in the compound database 11, a reaction in a compound pair for which a certain compound (for example, compound D) is not known to be synthesized from a certain compound (for example, compound C) is expressed as an unknown reaction. To do. For a compound stored in the compound database 11, a compound pair for which a certain compound (for example, compound D) is not known to be synthesized from a certain compound (for example, compound C) is referred to as an unknown compound pair. .

<Known reaction database 12>
In the known reaction database 12, the names of the respective compounds in the known compound pairs, the reaction directions in the known compound pairs, and the known reaction enzymes are stored in association with each other. There may be a plurality of known reaction enzymes.

For example, the name of each compound in the compound pair registered in the KEGG database, the reaction direction in the reaction of the compound pair, and the enzyme used in the reaction in the compound pair may be associated and stored in the known reaction database 12. Good. In addition, for example, the name of each compound in a compound pair registered in a metabolite database such as PubChem, BRENDA, MetaCyc, the reaction direction in the compound pair, and the enzyme used for the reaction in the compound pair are related to each other in the known reaction It may be stored in the database 12. Alternatively, the name of each compound in the compound pair registered in the metabolic reaction database of E. coli, the reaction direction in the compound pair, and the enzyme used for the reaction in the compound pair may be associated and stored in the known reaction database 12. Good. Not limited to this, each compound name registered in various chemical substance databases, the reaction direction in the compound pair, and the enzyme used for the reaction in the compound pair are associated and stored in the known reaction database 12. May be.

<Partial structure database 13>
The partial structure database 13 stores partial data of the chemical structural formula of the compound and an identification number assigned to the partial data of the chemical structural formula of the compound. The partial data of the chemical structure of the compound is data created by extracting a partial chemical structure from the chemical structure of the compound according to a predetermined standard. For example, based on the atom types registered in the KEGG database, partial chemical structure formula partial data is created by extracting a partial chemical structure from the chemical structure formula of the compound registered in the compound database 11 May be. The identification number assigned to the partial data of the chemical structural formula of the compound is expressed as a structure number. For example, in the example of FIG. 2, C1a, C1b, C1c, and C1d correspond to structure numbers.

<Common structure database 14>
The common structure database 14 stores data obtained by grouping partial data of chemical structural formulas of compounds and identification numbers assigned to the grouped data. An identification number assigned to data obtained by grouping partial data of chemical structural formulas of compounds is referred to as a common structure number. For example, in the example of FIG. 2, C01 corresponds to the common structure number.

<UF database 15>
The UF database 15 stores the UF of a compound in association with the name of the compound.

<Creation of compound UF>
The control unit 21 creates a UF of the compound stored in the compound database 11. Then, the control unit 21 stores the UF of the created compound in the UF database 15 in association with the name of the compound.

  Note that the compound UF and the compound name stored in the UF database 15 are the same, but the compound names may be different. For example, it is assumed that Compound A and Compound B are classified using the example of FIG. 2, and for Compound A, there are 2 C1a, and for Compound B, there is 1 C1a and 1 C1b. Since C1a and C1b are grouped, the compound UF is created assuming that both compound A and compound B have two C01. Therefore, the UF of the compound is the same, but the name of the compound may be different. In this case, the names of a plurality of compounds are stored in the UF database 15 in association with the UF of the compound.

  In the above, the control unit 21 creates the UF of the compound with reference to the common structure database 14. That is, the control unit 21 creates a UF of a compound by referring to data obtained by grouping partial data of chemical structural formulas of the compound and a common structure number. However, the present invention is not limited to this, and the control unit 21 may create the UF of the compound with reference to the partial structure database 13. That is, the control unit 21 may create the UF of the compound by referring to the partial data of the chemical structural formula of the compound and the structure number.

<Difference database 16>
The difference database 16 stores the UF difference of the compound pair and the name of each compound in the compound pair in association with each other. For example, a value obtained by subtracting the UF of the compound B from the UF of the compound A is the difference of the UF of the compound pair. The difference database 16 may store the UF of each compound in the compound pair instead of the name of each compound in the compound pair.

<Create DF to be stored in difference database 16>
The control unit 21 creates pairs for all the compounds stored in the UF database 15 and calculates the UF difference of the created compound pairs. The difference in UF of a compound pair is expressed as DF (Difference Feature).

  The control unit 21 creates pairs for all the compounds stored in the UF database 15 on the assumption that reactions occur in all the compound pairs stored in the UF database 15. For example, when the compounds A, B, and C are stored in the UF database 15, the control unit 21 performs a pair of the compound A and the compound B, a pair of the compound A and the compound C, and a pair of the compound B and the compound C. Create Further, for example, when calculating the DF for the pair of compound A and compound B, the control unit 21 calculates the value obtained by subtracting the UF of compound B from the UF of compound A, and the UF of compound A from the UF of compound B. The subtracted value is calculated. Then, the control unit 21 stores the DF and the name of each compound in the compound pair in the difference database 16 in association with each other.

<Difference database 17>
In the difference database 17, the UF difference of the known compound pair, the name of each compound in the known compound pair, and the known reaction enzyme are stored in association with each other. The difference database 17 may store the UF of each compound in the known compound pair instead of the name of each compound in the known compound pair.

<Create DF to be stored in difference database 17>
The control unit 21 calculates the UF difference of a known compound pair among the compounds stored in the UF database 15. The UF difference of a known compound pair is denoted as known DF. For example, when the reaction for synthesizing the compound B from the compound A is a known reaction, the control unit 21 calculates the known DF by subtracting the UF of the compound A from the UF of the compound B. Then, the control unit 21 stores the known DF, the name of each compound in the known compound pair, and the known reaction enzyme in the difference database 17 in association with each other. The control unit 21 uses the data stored in the known reaction database 12 for the names and known reaction enzymes of each compound in the known compound pair.

<Creation of compound synthesis route by virtual reaction method>
With reference to FIG. 6, the flow of creation of a compound synthesis route by a virtual reaction method will be described. The flow in FIG. 6 is started by receiving an instruction to create a compound synthesis route by a virtual reaction method from the user via the operation unit 10. At this time, the designation of the starting compound and the target compound may be received from the user via the operation unit 10.

  In the process of S601 in FIG. 6, the control unit 21 creates a UF of a compound stored in the compound database 11. That is, the control unit 21 counts the number of partial structures included in the chemical structure of the compound stored in the compound database 11 for each type of partial structure, and includes the chemical structure in the compound. The chemical structural formula of the compound is converted into a numerical value by counting the number of bonded states of the partial structure for each type of partial structure and bonded state. In the process of S601 in FIG. 6, the control unit 21 stores the created UF and the compound name in the UF database 15 in association with each other. The control unit 21 that executes the process of S601 in FIG. 6 functions as a conversion unit.

  Next, in the process of S602 in FIG. 6, the control unit 21 creates pairs for all the compounds stored in the UF database 15, and calculates the DF of the created compound pair. The control unit 21 stores the calculated DF and the name of each compound in the compound pair in the difference database 16 in association with each other.

  In the process of S602 in FIG. 6, the control unit 21 calculates a known DF for a known compound pair among the compound pairs stored in the UF database 15. The control unit 21 stores the calculated known DF, the name of each compound in the known compound pair, and the known reaction enzyme in the difference database 17 in association with each other. The control unit 21 that executes the process of S602 in FIG. 6 functions as a calculation unit.

  In the process of S603 in FIG. 6, the control unit 21 compares the DF stored in the difference database 16 with the known DF stored in the difference database 17.

  Next, in the process of S604 in FIG. 6, the control unit 21 performs an unknown reaction in an unknown compound pair for a DF that matches the known DF stored in the difference database 17 among the DFs stored in the difference database 16. Determine as a virtual response. It is expected that an unknown reaction in an unknown compound pair for a DF that matches the known DF can occur. Note that the control unit 21 does not determine a known reaction in a known compound pair for a DF that matches the known DF stored in the difference database 17 among the DFs stored in the difference database 16 as a virtual reaction.

  In the process of S604 in FIG. 6, when the DF stored in the difference database 16 and the known DF stored in the difference database 17 match, the control unit 21 is associated with the matching known DF. The known reaction enzyme is determined as the enzyme used for the virtual reaction. When there are a plurality of known reaction enzymes associated with the known DF, the control unit 21 determines the plurality of known reaction enzymes as enzymes used for the virtual reaction. The control unit 21 that executes the processes of S603 and S604 in FIG. 6 functions as a determination unit.

  Next, in the process of S605 of FIG. 6, the control unit 21 associates the name of each compound of the compound pair regarding the virtual reaction and the enzyme used for the virtual reaction, and stores them in the virtual reaction database 18.

  6, the control unit 21 creates a compound synthesis route based on the known reaction database 12 and the virtual reaction database 18. In this case, the control unit 21 uses a node (vertex) indicating each compound of the compound pair and each compound of the compound pair related to the virtual reaction, and an edge (side) indicating the known reaction and the virtual reaction as a compound synthesis route. Create That is, the control unit 21 constructs a graph (network) including all reactions by using each compound of the known compound pair and each compound of the compound pair related to the virtual reaction as a node, and using the known reaction and the virtual reaction as an edge. Create synthetic pathways for compounds. The control unit 21 refers to the known reaction database 12 for each compound of the known compound pair and the known reaction, and refers to the virtual reaction database 18 for each compound and the virtual reaction of the compound pair regarding the virtual reaction. The control unit 21 that executes the process of S606 in FIG. 6 functions as a creation unit.

  The graph is formally defined as an ordered pair G = (V, E) consisting of a set of nodes V and a set of edges E connecting the nodes. The order pair G represents how the compounds included in the graph are adjacent (form a network) through each reaction. The graph may be expressed by a graphic or may be expressed by an array process using each element of the array as a node.

  FIG. 7 shows an example in which the graph is represented by a graphic. In FIG. 7, each compound in the compound pair with respect to each compound and virtual reaction of the known compound pair is indicated by nodes 201-207, and the known reaction and virtual reaction are indicated by edges 301-307. Moreover, in FIG. 7, the solid line edge shows the known reaction, and the broken line edge shows the virtual reaction.

  Next, in the process of S607 in FIG. 6, the control unit 21 searches for the synthesis route from the starting compound to the target compound from the compound synthesis route created in the process of S606 in FIG. The control unit 21 may search for a synthetic route from the starting compound to the target compound including a predetermined number of reaction steps or less from the synthetic route of the compound created in the process of S606 in FIG. The control unit 21 may search the synthesis route from the starting compound to the target compound from the compound synthesis route created in the process of S606 of FIG. 6 by depth-first search or breadth-first search. The control unit 21 uses a starting compound or a raw material compound as a starting point and a target compound as an end point (forward search method), or a method for performing a route search using a target compound as a starting point and the starting compound or raw material compound as an end point ( (Reverse direction search) may be performed. The raw material compound is a compound that becomes a raw material when the target compound is synthesized. Depending on the purpose of the route search, it may be determined whether to perform the forward search method or the backward search method. You may perform designation | designated of a raw material compound by receiving from a user via the operation part 10. FIG.

  Next, in the process of S608 in FIG. 6, the control unit 21 selects the synthesis route from the starting compound to the target compound from the compound synthesis route created in the process of S606 in FIG. 6 (procedure 1). In the process of S608 in FIG. 6, instead of the procedure 1, the control unit 21 creates a synthesis route from the starting compound to the target compound including a predetermined number of reaction steps or less by the compound created in the process of S606 in FIG. (Procedure 2). In the process of S608 in FIG. 6, instead of the procedure 1, the control unit 21 performs the synthesis route from the starting compound to the target compound including the predetermined number or the predetermined range of reaction steps in the process of S606 in FIG. You may choose from the synthetic route of the created compound (procedure 3). The control unit 21 that executes the process of S608 in FIG. 6 functions as a selection unit.

  For example, in FIG. 7, when the node 201 is a raw material compound, the node 203 is a starting compound, and the node 205 is a target compound, a synthesis path including nodes 203, 204, and 205 and edges 303 and 304, and a node A synthetic route including 203, 207, and 205 and edges 306 and 307 is selected as a synthetic route from the starting compound to the target compound. Nodes 204 and 207 are intermediate compounds.

  In the process of S609 in FIG. 6, the control unit 21 starts from the starting compound including a predetermined number or less of virtual reaction steps in the synthetic route from the starting compound selected in the process of S608 in FIG. 6 to the target compound. A synthetic route to the target compound is extracted (procedure A). In the process of S609 in FIG. 6, instead of the procedure A, the control unit 21 has a predetermined number or a predetermined number of ranges in the synthesis route from the starting compound selected in the process of S608 in FIG. 6 to the target compound. A synthetic route from the starting compound including the virtual reaction step to the target compound may be extracted (procedure B).

  Next, in the process of S610 in FIG. 6, the control unit 21 determines the thermodynamic feasibility of the synthesis route from the starting compound extracted in the process of S609 in FIG. 6 to the target compound.

  The determination of thermodynamic feasibility is based on the change in free energy in each reaction included in the synthesis route from the starting compound to the target compound extracted in the process of S609 in FIG. 6, for example, Biophys. J. Vol. , No.7, pp1792-1805 (2007), and the direction of each reaction is determined based on the calculation result. When each reaction included in the synthesis route from the starting compound to the target compound extracted in the process of S609 in FIG. 6 is a forward reaction and a reversible reaction, the control unit 21 extracts in the process of S609 in FIG. It is determined that there is a thermodynamic feasibility for the synthetic route from the determined starting compound to the target compound. Further, when there is a reverse reaction in each reaction included in the synthesis route from the starting compound extracted in the process of S609 in FIG. 6 to the target compound, the control unit 21 starts the process extracted in the process of S609 in FIG. It is determined that there is no thermodynamic feasibility for the synthetic route from the compound to the target compound.

  Next, in the process of S611 in FIG. 6, the control unit 21 selects a thermodynamically feasible synthesis path from among the synthesis paths from the starting compound extracted in the process of S609 in FIG. 6 to the target compound. Extract. The control unit 21 that executes the processes of S610 and S611 in FIG. 6 functions as a thermodynamic determination unit.

  In the process of S612 in FIG. 6, the control unit 21 calculates the theoretical molar yield (%) of the target compound synthesized by the synthesis route extracted in the process of S611 in FIG. In this case, the control unit 21 may calculate the theoretical molar yield (%) from the raw material compound for the target compound synthesized by the synthesis route extracted in the process of S611 in FIG. When calculating the molar theoretical yield (%) from the raw material compound for the target compound synthesized by the synthesis route extracted in the process of S611 in FIG. 6, the control unit 21 refers to the known reaction database 12, A synthetic route from the starting compound to the starting compound is created. Then, the control unit 21 uses the synthesis path extracted from the process of S611 in FIG. 6 using the synthesis path from the raw material compound to the starting compound and the synthesis path extracted in the process of S611 in FIG. For the compound, the theoretical molar yield (%) from the raw material compound is calculated. You may perform designation | designated of a raw material compound by receiving from a user via the operation part 10. FIG. In this case, the control unit 21 may create a synthesis route from the raw material compound to the starting compound with reference to data on the metabolic reaction of E. coli stored in the known reaction database 12. The calculation of the molar yield can be performed, for example, by the method described in Biotechnology and Bioengineering, Volume 42, p.59-73 (1993).

  Next, in the process of S613 in FIG. 6, the control unit 21 determines whether or not the theoretical molar yield (%) calculated in the process of S612 in FIG. 6 is greater than or equal to the threshold (or greater than the threshold). . For example, an arbitrary value such as 0%, 100%, or 200% can be set as the threshold value.

  Next, in the process of S614 in FIG. 6, the control unit 21 uses the molar theoretical yield (%) calculated in the process of S612 in FIG. 6 among the synthesis paths extracted in the process of S611 in FIG. A synthesis route for synthesizing the target compound which is the above (or larger than the threshold value) is extracted. The control unit 21 that executes the processes of S612, S613, and S614 in FIG. 6 functions as a molar theoretical yield determination unit.

  Then, in the process of S615 in FIG. 6, the control unit 21 extracts a practical synthesis path for the synthesis path extracted in the process of S614 in FIG. Practical synthesis pathways may be extracted by taking into account changes in the number of carbon atoms in each reaction, the size of the carbon number, and the difficulty of modifying individual virtual reaction enzymes. .

In addition, the control unit 21 converts the virtual reaction in the synthesis route from the starting compound selected in the processing of S608 in FIG. 6 to the target compound into an enzymatic reaction that has been proven with reference to information obtained in advance. You may make it limit beforehand. Furthermore, the control unit 21 may narrow down the synthesis route by evaluating whether or not a virtual reaction is realized in the synthesis route from the starting compound selected in the processing of S608 of FIG. 6 to the target compound. In this case, the comparison of the partial structures of the compounds may be taken in and the presence / absence of the virtual reaction may be evaluated.

  Note that the control unit 21 may omit the process of S609 in FIG. 6, may omit the processes of S610 and S611 in FIG. 6, or may omit the processes of S612 to S614 in FIG. Alternatively, the process of S615 in FIG. 6 may be omitted. Further, the control unit 21 changes the order in which the processing of S609 in FIG. 6, the processing of S610 and S611 in FIG. 6, the processing from S612 to S614 in FIG. 6, and the processing in S615 in FIG. May be. For example, the control unit 21 may execute the process of S609 of FIG. 6 after executing the processes of S610 and S611 of FIG.

  Next, in the process of S616 in FIG. 6, the control unit 21 extracts the synthesis route extracted in the process of S615 in FIG. 6, the names of the starting compound, the intermediate compound, and the target compound included in the synthesis route, and the synthesis route. The information including the enzyme used in each reaction in is output. In this case, the control unit 21 specifies whether each reaction in the synthesis route extracted in the process of S615 of FIG. 6 is a known reaction or a virtual reaction, and is extracted in the process of S615 of FIG. The information of the synthesized route is output. The controller 21 may use an enzyme EC number as an enzyme used in each reaction in the synthesis pathway.

  For example, the control unit 21 may output information on the synthesis route extracted in the process of S615 of FIG. 6 using the graph 80 and the table 81 illustrated in FIG. As shown in FIG. 8, it is specified that the reaction for synthesizing the intermediate compound (compound B) from the starting compound (compound A) is a known reaction, and the target compound (compound C) is synthesized from the intermediate compound (compound B). It has been identified that the reaction is a virtual reaction. In FIG. 8, known reaction enzymes A and B are assigned as enzymes used for known reactions, and known reaction enzymes B and C are assigned as enzymes used for virtual reactions.

  The control unit 21 may rank the synthesis paths extracted in the process of S614 in FIG. 6 in descending order of the theoretical molar yield (%) calculated in the process of S612 in FIG. Furthermore, the control unit 21 provides information on the synthesis routes that are ranked in descending order of the theoretical molar yield (%) calculated in the process of S612 of FIG. 6 to the information output in the process of S616 of FIG. May be added.

  The control unit 21 may store the information output in the process of S616 in FIG. Further, the information output in the process of S616 in FIG. 6 may be displayed on the display unit 22.

  The control unit 21 converts the chemical structure of the compound into UF according to a predetermined standard, and calculates the DF by subtracting the UF for the other compound from the UF for one compound of the compound pair. The control unit 21 compares the known DF of the known compound pair with the DF of the unknown compound pair, and determines the unknown reaction of the unknown compound pair for the DF that matches the known DF of the known compound pair as a virtual reaction. The control unit 21 creates a compound synthesis route by using the nodes indicating the compounds of the known compound pair and the nodes indicating the compounds of the compound pair regarding the virtual reaction and the edges indicating the known reaction and the virtual reaction. The control unit 21 includes a synthesis route from the starting compound to the target compound, including each compound of the known compound pair and each compound of the compound pair regarding the virtual reaction, and an edge indicating the known reaction and the virtual reaction. Select from the synthetic route of the compound. It is possible to select a synthesis route of a new compound from a starting compound to a target compound from a synthesis route of a compound created by a node indicating a compound and an edge indicating a known reaction and a virtual reaction.

The control unit 21 compares the known DF of the known compound pair with the DF of the unknown compound pair, and converts the known reaction enzyme associated with the known DF of the known compound pair that matches the DF of the unknown compound pair to the virtual reaction. Determine as the enzyme used. It becomes possible to use a known reaction enzyme for the virtual reaction included in the synthetic route of the compound from the selected starting compound to the target compound.

<Creation of compound synthesis route by optimization method>
With reference to FIG. 9, the flow of creation of the compound synthesis route by the optimization method will be described. The flow in FIG. 9 is started by receiving an instruction to create a compound synthesis route by an optimization method from the user via the operation unit 10. At this time, the designation of the starting compound and the target compound may be received from the user via the operation unit 10.

  In the process of S901 in FIG. 9, the control unit 21 creates a UF of a compound stored in the compound database 11. That is, the control unit 21 counts the number of partial structures included in the chemical structure of the compound stored in the compound database 11 for each type of partial structure, and includes the chemical structure in the compound. The chemical structural formula of the compound is converted into a numerical value by counting the number of bonded states of the partial structure for each type of partial structure and bonded state. In the process of S901 in FIG. 9, the control unit 21 stores the created UF and the compound name in the UF database 15 in association with each other. The control unit 21 that executes the process of S901 in FIG. 9 functions as a conversion unit.

  Next, in the process of S902 in FIG. 9, the control unit 21 calculates a known DF for a known compound pair among the compound pairs stored in the UF database 15. The control unit 21 stores the calculated known DF, the name of each compound in the known compound pair, and the known reaction enzyme in the difference database 17 in association with each other. The control unit 21 that executes the process of S902 in FIG. 9 functions as a first calculation unit.

  In the process of S903 in FIG. 9, the control unit 21 calculates the difference between the UF of the compound pair of the starting compound and the target compound by subtracting the UF for the starting compound from the UF for the target compound. The difference in UF of the compound pair between the starting compound and the target compound is expressed as Difference Total. The designation of the starting compound and the target compound is performed by receiving from the user via the operation unit 10. The control unit 21 that executes the process of S903 in FIG. 9 functions as a second calculation unit.

  Next, in the processing of S904 in FIG. 9, the control unit 21 uses the integer programming to extract the plurality of known DFs from the difference database 17 so that the total value of the plurality of known DFs matches the value of Difference Total. Extract. In this case, the control unit 21 may limit the number of known DFs and extract a plurality of known DFs from the difference database 17. For example, the control unit 21 may extract a predetermined number or less or a predetermined number of known DFs from the difference database 17. By specifying an appropriate condition using integer programming, it is possible to perform a search from a wide range of combinations in a short calculation time. However, the control unit 21 may use another method instead of the integer programming.

  The control unit 21 creates a set (group) including a plurality of known DFs by repeating the process of S904 in FIG. 9 a predetermined number of times. A set including a plurality of known DFs is referred to as a DF combination group. The control unit 21 creates one or more types of DF combination groups by randomly extracting a plurality of known DFs from the difference database 17 each time the processing of S904 in FIG. 9 is repeated.

In the process of S904 in FIG. 9, the control unit 21 may extract a plurality of known DFs from the difference database 17 and create a DF combination group using the extracted plurality of known DFs. And the control part 21 repeats the process of S904 of FIG.
You may make it produce the type or multiple types of DF combination group. In this case, the control unit 21 may randomly extract a plurality of known DFs from the difference database 17 each time the process of S904 in FIG. 9 is repeated.

  Next, in the process of S905 in FIG. 9, the control unit 21 uses the starting compound, the target compound, and a plurality of known DFs included in the DF combination group for the DF combination group created in the process of S904 in FIG. Create synthetic pathways for compounds. When a plurality of types of DF combination groups are created in the process of S904 in FIG. 9, the control unit 21 creates a compound synthesis route for each DF combination group. The control unit 21 that executes the processes of S904 and S905 in FIG. 9 functions as a creation unit.

  FIG. 10 shows an example of a synthetic route of a compound composed of a starting compound and a target compound and a plurality of known DFs included in the DF combination group. As shown in FIG. 10, there are six combinations of a plurality of known DFs included in the DF combination group.

  In the process of S906 in FIG. 9, the control unit 21 calculates the UF of the intermediate compound included in the compound synthesis route created in the process of S905 in FIG. The control unit 21 calculates the UF of the intermediate compound according to the following procedure.

  As an example, the synthesis route of the compound “starting compound, known DF1, known DF2, known DF3, target compound” in the DF combination group of FIG. 10 will be described. Further, the case where the intermediate compound A is synthesized from the starting compound, the intermediate compound B is synthesized from the intermediate compound A, and the target compound is synthesized from the intermediate compound B will be described. First, the control unit 21 calculates the UF of the intermediate compound B by subtracting the known DF3 from the UF of the target compound. Next, the control unit 21 calculates the UF of the intermediate compound A by subtracting the known DF2 from the UF of the intermediate compound B. In this way, the control unit 21 calculates the UF of the intermediate compound included in the compound synthesis route created by the processing of S905 of FIG. 9 by sequentially subtracting each known DF from the UF of the target compound.

  In the process of S906 in FIG. 9, the control unit 21 sets the compound created in the process of S905 in FIG. 9 for all combinations of a plurality of known DFs included in the DF combination group created in the process of S904 in FIG. 9. The UF of the intermediate compound included in the synthesis route is calculated. When a plurality of types of DF combination groups are created in the process of S904 in FIG. 9, the control unit 21 calculates the UF of the intermediate compound included in the compound synthesis route for each DF combination group. The control unit 21 that executes the process of S906 in FIG. 9 functions as a third calculation unit.

  Next, in the process of S907 in FIG. 9, the control unit 21 determines whether or not the synthetic route of the compound created in the process of S905 in FIG. 9 is established as a synthetic route from the starting compound to the target compound. In the case where a plurality of compound synthesis routes have been created in the process of S905 of FIG. 9, the control unit 21 determines whether each synthesis route is established as a synthesis route from the starting compound to the target compound.

An example of determining whether or not the synthetic route from the starting compound to the target compound is established will be described.
[Judgment Example 1] When the UF of the intermediate compound included in the compound synthesis route created in the process of S905 of FIG. 9 has a negative value, the control unit 21 determines the compound synthesis route including the intermediate compound. The synthetic route from the starting compound to the target compound is determined not to be established. When the UF of the intermediate compound has a negative value, the intermediate compound is not established as a compound, so the control unit 21 establishes the synthesis route of the compound including the intermediate compound as a synthesis route from the starting compound to the target compound. Judge that not.

  [Judgment Example 2] The control unit 21 compares the UF atomic information of the intermediate compound included in the compound synthesis route created by the processing of S905 of FIG. 9 with the bond information. If there is a discrepancy between the UF atomic information and the bond information of the intermediate compound included in the compound synthesis route created in the process of S905 in FIG. The synthesis route is determined not to be established as a synthesis route from the starting compound to the target compound. That is, if the number of partial structures included in the chemical structure of the intermediate compound and the number of bonding states of the partial structures included in the chemical structure of the intermediate compound do not match, the control unit 21 It is determined that the synthesis route of the compound including the compound is not established as the synthesis route of the compound from the starting compound to the target compound.

  Next, in the processing of S908 in FIG. 9, the control unit 21 uses the synthesis route from the starting compound to the target compound in the processing of S907 in FIG. 9 among the synthesis routes of the compound created in the processing of S905 in FIG. The synthesis route of the compound determined to be established is extracted.

  In the process of S909 in FIG. 9, the control unit 21 determines the enzyme used in the synthesis of the compound included in the compound synthesis path for the compound synthesis path extracted in the process of S908 in FIG. In this case, the control unit 21 refers to the difference database 17 to determine an enzyme used in the synthesis of the compound included in the compound synthesis pathway. In the difference database 17, the known DF and the known reaction enzyme are stored in association with each other. Therefore, the control unit 21 assigns the known reaction enzyme stored in association with the known DF stored in the difference database 17 to each known DF included in the compound synthesis pathway extracted in the process of S908 of FIG. By assigning to each other, the enzyme used in the synthesis of the compound included in the compound synthesis pathway is determined. The control unit 21 may assign a plurality of known reaction enzymes stored in the difference database 17 to each known DF included in the compound synthesis route extracted in the process of S908 of FIG.

  Next, in the process of S910 in FIG. 9, the control unit 21 determines, for each of the compound synthesis pathways extracted in the process of S908 in FIG. 9, based on the UF of the intermediate compound included in the compound synthesis pathway. The name of the intermediate compound included in the synthetic route is determined. In this case, the control unit 21 refers to the UF database 15 and determines the name of the intermediate compound included in the compound synthesis route. When the UF of the intermediate compound included in the compound synthesis route matches the UF of the compound stored in the UF database 15, the control unit 21 associates with the UF of the compound stored in the UF database 15. The name of the existing compound is determined as the name of the intermediate compound included in the compound synthesis route. When the names of a plurality of compounds are associated with the UF of the compound and stored in the UF database 15, the control unit 21 determines a plurality of names for the intermediate compound included in the compound synthesis route.

  When the UF of the intermediate compound included in the compound synthesis route and the UF of the compound stored in the UF database 15 do not match, the control unit 21 does not determine the name of the intermediate compound included in the compound synthesis route. A predetermined number is assigned to the intermediate compound. An intermediate compound to which a predetermined number is assigned is referred to as a virtual compound without determining the name of the intermediate compound included in the compound synthesis route.

  For example, the compound database 11 may store the name of the compound registered in the KEGG database and the data of the chemical structural formula of the compound in association with each other. In this case, the UF database 15 stores only the UF of the compound registered in the KEGG database. When only the UF of the compound registered in the KEGG database is stored in the UF database 15, the UF of the intermediate compound included in the compound synthesis route matches the UF of the compound stored in the UF database 15. There is a possibility not to.

  In the process of S910 in FIG. 9, the control unit 21 determines whether each reaction included in the compound synthesis path is a known reaction for each of the compound synthesis paths extracted in the process of S908 in FIG. Determine if it is a reaction. Whether each reaction included in the synthesis route of the compound is a known reaction or a virtual reaction is determined by the following process.

  The control unit 21 extracts a known compound pair associated with the known DF of the intermediate compound included in the compound synthesis route extracted in the process of S908 of FIG. 9 from the difference database 17. When the names of the compounds in the extracted known compound pair match the names of the compounds in the compound pair included in the compound synthesis route extracted in the process of S908 in FIG. 9, the control unit 21 matches the compound. The paired response is determined to be a known response. If the names of the compounds in the extracted known compound pair do not match the names of the compounds in the compound pair included in the compound synthesis route extracted in the process of S908 in FIG. The pair response is determined as a virtual response. In addition, when the virtual compound is included in the compound synthesis route extracted in the process of S908 in FIG. 9, the control unit 21 synthesizes another compound from the virtual compound and the reaction that synthesizes the virtual compound. Is determined as a virtual reaction.

  Next, in the process of S911 in FIG. 9, the control unit 21 determines the thermodynamic feasibility of the synthesis route to the compound extracted in the process of S908 in FIG.

  The determination of thermodynamic feasibility is performed by calculating the free energy change in each reaction included in the synthesis route of the compound extracted in the process of S908 in FIG. 9, and determining the direction of each reaction based on the calculation result. Is done. When the reactions included in the synthesis route of the compound extracted in the process of S908 in FIG. 9 are a forward reaction and a reversible reaction, the control unit 21 synthesizes the compound extracted in the process of S908 in FIG. Determine that the path has thermodynamic feasibility. In addition, when there is a reverse reaction in each reaction included in the compound synthesis route extracted in the process of S908 in FIG. 9, the control unit 21 heats the compound synthesis route extracted in the process of S908 in FIG. Determine that there is no dynamic feasibility.

  Then, in the process of S912 in FIG. 9, the control unit 21 extracts a synthesis path that is thermodynamically feasible from the compound synthesis paths extracted in the process of S908 in FIG. The control unit 21 that executes the processes of S911 and S912 in FIG. 9 functions as a thermodynamic determination unit.

  When the virtual compound is included in the compound synthesis route extracted in the process of S908 of FIG. 9, the control unit 21 executes the processes of S911 and S912 of FIG. 9 after determining the name of the virtual compound. The control unit 21 may determine the name of the virtual compound by referring to a database provided in a device on the external network. For example, when only the UF of a compound registered in the KEGG database is stored in the UF database 15, the control unit 21 refers to a metabolite database such as PubChem, BRENDA, MetaCyc, etc. You may decide. Further, the control unit 21 inquires the user of the name of the virtual compound via the display unit 22 and determines the name of the virtual compound by receiving an input of the name of the virtual compound from the user via the operation unit 10. Good.

It is necessary to grasp the chemical structure of a compound to determine whether it is a thermodynamically feasible synthetic route. Therefore, the control unit 21 determines the name of the compound included in the compound synthesis route extracted in the processing of S908 in FIG. 9, and grasps the chemical structure of the compound from the name of the compound, and can be executed thermodynamically. Judging the synthetic route The control unit 21 may grasp the chemical structure of the compound by referring to the compound database 11. The control part 21 may grasp | ascertain the chemical structure of a compound by referring the database with which the apparatus on an external network is provided. The control unit 21 may grasp the chemical structure of the compound by inquiring of the chemical structure of the compound via the display unit 22 and accepting input of chemical structure data of the compound from the user via the operation unit 10. .

  Next, in the process of S913 in FIG. 9, the control unit 21 calculates the molar theoretical yield (%) of the target compound synthesized by the synthesis route extracted in the process of S912 in FIG. In this case, the control unit 21 may calculate the theoretical molar yield (%) from the raw material compound for the target compound synthesized by the synthesis route extracted in the process of S912 in FIG. When calculating the theoretical molar yield (%) from the raw material compound for the target compound synthesized by the synthesis route extracted in the process of S912 in FIG. 9, the control unit 21 refers to the known reaction database 12, A synthetic route from the starting compound to the starting compound is created. Then, the control unit 21 uses the synthesis route from the raw material compound to the starting compound and the synthesis route extracted in the processing of S912 in FIG. 9 to synthesize by the synthesis route extracted in the processing of S911 in FIG. For the compound, the theoretical molar yield (%) from the raw material compound is calculated. You may perform designation | designated of a raw material compound by receiving from a user via the operation part 10. FIG. In this case, the control unit 21 may create a synthesis route from the raw material compound to the starting compound with reference to data on the metabolic reaction of E. coli stored in the known reaction database 12. The calculation of the molar yield can be performed, for example, by the method described in Biotechnology and Bioengineering, Volume 42, p.59-73 (1993).

  Next, in the process of S914 in FIG. 9, the control unit 21 determines whether or not the theoretical molar yield (%) calculated in the process of S913 in FIG. 9 is equal to or greater than a threshold (or greater than the threshold). To do. For example, an arbitrary value such as 0%, 100%, or 200% can be set as the threshold value.

  Then, in the process of S915 of FIG. 9, the control unit 21 has the theoretical molar yield (%) calculated by the process of S913 of FIG. 9 among the synthesis paths extracted by the process of S912 of FIG. A synthesis route of a compound for synthesizing a target compound that is (or larger than a threshold value) is extracted. The control unit 21 that executes the processes of S913, S914, and S915 in FIG. 9 functions as a molar theoretical yield determination unit. Even when a virtual compound is included in the compound synthesis route extracted in the process of S908 of FIG. 9, the control unit 21 can execute the processes of S913, S914, and S915 of FIG. is there. Even when a conversion reaction using an unknown virtual compound as a substrate or product is included in the synthesis route, the number of partial structures (atoms) constituting the virtual compound is included in the UF of the virtual compound. Change (increase / decrease in the number of molecules) can be grasped.

  Next, in the process of S916 in FIG. 9, the control unit 21 extracts a practical synthesis path for the synthesis path extracted in the process of S915 in FIG. Practical synthesis pathways may be extracted by taking into account changes in the number of carbon atoms in each reaction, the size of the carbon number, and the difficulty of modifying individual virtual reaction enzymes. .

  Note that the control unit 21 may omit the processes of S911 and S912 in FIG. 9, may omit the processes of S913 to S915 of FIG. 9, or may omit the process of S916 of FIG. Good. Moreover, the control part 21 may change the order which performs the process of S911 and S912 of FIG. 9, the process of S913 to S915 of FIG. 9, and the process of S916 of FIG. For example, the control unit 21 may execute the processes of S911 and S912 of FIG. 9 after executing the processes of S913 to S915 of FIG.

Next, in the process of S917 in FIG. 9, the control unit 21 extracts the synthesis route extracted in the process of S916 in FIG. 9, the names of the starting compound, the intermediate compound, the target compound, and the UFs included in the synthesis route. And information including enzymes used in each reaction in the synthetic pathway. In this case, the control unit 21 specifies whether each reaction in the synthesis route extracted in the process of S916 in FIG. 9 is a known reaction or a virtual reaction, and is extracted in the process of S916 in FIG. The information of the synthesized route is output. The controller 21 may use an enzyme EC number as an enzyme used in each reaction in the synthesis pathway. When the virtual compound is included in the synthesis route extracted in the process of S916 in FIG. 9, the control unit 21 replaces the name of the intermediate compound with the predetermined number assigned to the intermediate compound, and displays the predetermined number in S917 in FIG. It is added to the information output by the process.

  For example, the control unit 21 uses the tables 121 and 122 shown in FIG. 11 to extract the synthesis route extracted in the process of S916 of FIG. 9, the names of the starting compound, intermediate compound, and target compound included in the synthesis route, and the UF. And an enzyme used in each reaction in the synthesis route. FIG. 11 is an example when two synthesis paths are output.

In Table 121 and Table 122, compounds A, B, C and D are the names of the compounds, VC00001
Is a predetermined number assigned to the virtual compound. In Table 121, it is specified that the reaction for synthesizing the intermediate compound A from the starting compound and the reaction for synthesizing the intermediate compound B from the intermediate compound A are known reactions. In Table 121, it is specified that the reaction for synthesizing the target compound from the intermediate compound B is a virtual reaction. In Table 122, it is specified that the reaction for synthesizing the intermediate compound A from the starting compound is a known reaction. Further, in Table 122, it is specified that the reaction for synthesizing the intermediate compound C from the intermediate compound A and the reaction for synthesizing the target compound from the intermediate compound C are virtual reactions.

  The known reaction enzymes A and B in Tables 121 and 122 are enzymes used for the reaction of a pair of a compound specified by UF3 and a compound specified by UF4. Known reaction enzymes C and D in Tables 121 and 121 are enzymes used for the reaction of a pair of a compound specified by UF5 and a compound specified by UF6. The known DF1s in Tables 121 and 122 have two patterns: a value obtained by subtracting UF4 from UF3 and a value obtained by subtracting UF6 from UF5. That is, the value obtained by subtracting UF4 from UF3 and the value obtained by subtracting UF6 from UF5 are the same value. Therefore, in Tables 121 and 122, known reaction enzymes A, B, C, and D are assigned as enzymes used in the reaction for synthesizing intermediate compound A from the starting compound.

  Known reaction enzymes E and F in Table 121 are enzymes used for the reaction in a pair of a compound specified by UF7 and a compound specified by UF8. The known DF2 in Table 121 is a value obtained by subtracting UF8 from UF7. Therefore, in Table 121, known reaction enzymes E and F are assigned as enzymes used in the reaction of synthesizing intermediate compound B from intermediate compound A.

  Known reaction enzymes G and H in Table 121 are enzymes used for the reaction of a pair of a compound specified by UF9 and a compound specified by UF10. Known reaction enzymes I and J are enzymes used for the reaction of a pair of a compound specified by UF11 and a compound specified by UF12. The known DF3 in Table 121 has two patterns: a value obtained by subtracting UF10 from UF9 and a value obtained by subtracting UF12 from UF11. That is, the value obtained by subtracting UF10 from UF9 and the value obtained by subtracting UF12 from UF11 are the same value. Therefore, in Table 121, the known reaction enzymes G, H, I, and J are assigned as enzymes used in the reaction for synthesizing the target compound from the intermediate compound B.

  The known reaction enzymes K and L in Table 122 are enzymes used for the reaction in a pair of a compound specified by UF15 and a compound specified by UF16. The known DF4 in Table 122 is a value obtained by subtracting UF16 from UF15. Therefore, in Table 122, known reaction enzymes K and L are assigned as enzymes used in the reaction for synthesizing intermediate compound C from intermediate compound A.

  Known reaction enzymes M and N in Table 122 are enzymes used for the reaction of a pair of a compound specified by UF17 and a compound specified by UF18. Known reaction enzymes O and P are enzymes used for the reaction of a pair of a compound specified by UF19 and a compound specified by UF20. The known DF5 in Table 122 has two patterns: a value obtained by subtracting UF18 from UF17, and a value obtained by subtracting UF20 from UF19. That is, the value obtained by subtracting UF18 from UF17 and the value obtained by subtracting UF20 from UF19 are the same value. Therefore, in Table 122, the known reaction enzymes M, N, O, and P are assigned as enzymes used in the reaction for synthesizing the target compound from the intermediate compound C.

  The control unit 21 may rank the synthesis paths extracted in the process of S915 in FIG. 9 in descending order of the theoretical molar yield (%) calculated in the process of S913 in FIG. Further, the control unit 21 provides information on the synthesis routes that are ranked in descending order of the theoretical molar yield (%) calculated in the process of S913 in FIG. 9 to the information output in the process of S913 in FIG. May be added.

  The control unit 21 may store the information output in the process of S917 of FIG. Further, the control unit 21 may display the information output in the process of S917 in FIG.

  The control unit 21 converts the chemical structure of the compound into UF according to a predetermined standard, and calculates DF (first difference value) by subtracting the UF of the other compound from the UF of one compound of the compound pair. To do. The controller 21 calculates the DF (second difference value) of the pair of the starting compound and the target compound by subtracting the UF for the starting compound from the UF for the target compound. The control unit 21 creates a compound synthesis route so that the total value of the first difference values in the synthesis route from the starting compound to the target compound matches the second difference value. The compound synthesis route includes a starting compound, a target compound, and a plurality of first difference values.

  By creating a synthetic route for a compound composed of a starting compound and a target compound and a plurality of first difference values, it is possible to create a synthetic route for a new compound from the starting compound to the target compound. The compound included in the synthetic route from the prepared starting compound to the target compound may be a known compound or a virtual compound (unknown compound). Therefore, it is possible to create a synthetic route including a virtual compound. The reaction in the compound pair included in the synthetic route from the created starting compound to the target compound may be a known reaction or a virtual reaction (unknown reaction). Therefore, a synthetic route including a virtual reaction can be created.

  A known reaction enzyme is assigned to the first difference value included in the synthetic route from the created starting compound to the target compound. A known reaction enzyme can be used for a virtual reaction included in the synthesis route of the compound from the starting compound to the target compound.

  Examples will be described below. However, the embodiments of the present invention are not limited to the following examples.

1. Method for creating terephthalic acid (TPA) synthesis route using synthesis route creation program A specific example of creating a TPA synthesis route using a synthesis route creation program (described in the above reference example) will be described.
The name of the compound registered in the KEGG database and the chemical structure data of the compound, and the name of the target compound (including non-natural compounds) and the chemistry of the compound in addition to the compound registered in the KEGG database Using the structural formula data, the names of 13213 compounds and the chemical structural formula data of the compounds were stored in the compound database 11. By downloading the KCF format data represented by KEGG ATOM TYPE for all compounds in the KEGG database from the KEGG database, the compound name and the chemical structural data of the compound were stored in the compound database 11. Compounds not registered in the KEGG database were converted to KCF format using the KegDraw tool.
Under the above conditions, phenylalanine was designated as the starting compound, TPA was designated as the target compound, and creation of a compound synthesis route was started. The control unit 21 performs the process of S901 in FIG. 6, thereby creating a compound UF and storing it in the UF database 15. When the control unit 21 performs the process of S902 in FIG. 6, about 174 million known DFs are calculated and stored in the difference database 16.
When the control unit 21 performs the processes of S603 and S604 in FIG. 6, about 134,000 unknown reactions are determined as virtual reactions, and the control unit 21 performs the process of S605 in FIG. The control unit 21 performs the process of S606 in FIG. 6 to create a graph (network) including known reactions and virtual reactions.
When the control unit 21 performs the process of S607 in FIG. 6, the created synthetic route is searched by the forward search method using the starting compound as the start point and the target compound as the end point.
Under the condition that the synthesis route from the starting compound including four or less reaction steps to the target compound is selected, the control unit 21 performs the process of S608 in FIG. The synthetic route to the compound was selected. Under the condition that the synthesis route from the starting compound including the virtual reaction step of 3 or less to the target compound is extracted, the control unit 21 performs the processing of S609 in FIG. It was extracted as a synthetic route from the compound to the target compound.
The control unit 21 performs the processing of S610 and S611 in FIG. 6, so that 196 compound synthesis routes out of 7847 compound synthesis routes are extracted as thermodynamically feasible synthesis routes. . Glucose was designated as the raw material compound, and the control unit 21 performed the process of S612 in FIG. 6, thereby creating a synthesis route from the raw material compound to the starting compound. In this case, the control unit 21 refers to data on the metabolic reaction of E. coli stored in the known reaction database 12. Then, the control unit 21 performs the process of S612 in FIG. 6 to calculate the molar theoretical yield (%) from the raw material compound for the target compound synthesized by the synthesis route extracted in the process of S611 in FIG. It was done. Under the condition that glucose is designated as a raw material compound and a synthetic route of a compound for synthesizing a target compound having a theoretical molar yield (%) from the raw material compound of 0% or more is extracted, the control unit 21 performs S613 in FIG. , S614, and S615, the synthetic pathways of the two compounds were extracted as practical synthetic pathways (FIG. 12).

2. Method for creating p-toluic acid synthesis pathway using synthetic pathway creation program The search for the synthesis pathway for p-toluic acid was carried out in the same manner as in the above example except that the target compound was designated as p-toluic acid. . When the control unit 21 performs the process of S609 in FIG. 6, 4944 compound synthesis routes are extracted as synthesis routes from the starting compound to the target compound. As the control unit 21 performs the processing of S610 and S611 in FIG. 6, 493 compound synthesis routes out of 4944 compound synthesis routes are extracted as thermodynamically feasible synthesis routes. . Furthermore, when the control unit 21 performs the processing of S613, S614, and S615 in FIG. 6, a synthesis route of one compound is extracted as a practical synthesis route (FIG. 12).

3. Specific example 1 (R7-R3-R4) for preparing a microorganism that synthesizes terephthalic acid by a bio-method
E. coli strains capable of producing terephthalic acid can be obtained by general molecular biology (Ausubel e
t al, Current Protocol in Molecular Biology (2003); Sambrook et al, Molecular Cloning: By using the ^{A Laboratory Manual, 3 rd Edition (} 2001) , etc.), can be prepared by the following procedure.
An Anabaena variabilis-derived PAL gene (GenBank accession number FW572332.1) and Closrida bacterium-derived AbcDA operon (GenBank accession number: GU357991.1 GU357992.1) were ligated, and this ligated gene was ligated to the E. coli plasmid vector pUC19 (manufactured by Takara Bio Inc.). The plasmid pPAL-AbcDA incorporated into the downstream side of the lac promoter is constructed. Further, a recombinant E. coli strain carrying PAL and AbcDA is prepared by transforming E. coli JM109 strain with this plasmid DNA, and this E. coli strain is named JM109 / pPAL-AbcDA. The beta-oxidation reaction of R3 is carried out by a series of enzymes (fadBA) possessed by E. coli.
JM109 / pPAL-AbcDA is cultured by methods well known to those skilled in the art (Japan Biotechnology Society, edited by Biotechnology Experiments (2002); Vogel H. et al, Fermentation and Biochemical Engineering Handbook, 2nd Ed. (2007), etc.) be able to. For example, E. coli JM109 / pPAL-AbcDA is seeded in 100 ml of a minimal synthetic medium supplemented with 20 g / L glucose, 50 μg / ml ampicillin, 0.2 mM IPTG (isopropyl-β-D-thiogalactopyranoside), and the flask is By using it, it can culture | cultivate at 37 degreeC for 24 hours or more. When culturing using a jar fermenter, the substrate feed rate, the number of stirring revolutions, the aeration rate, the pH, and the like can be controlled within an appropriate range. An anaerobic condition, a microaerobic condition, and an aerobic condition can be selected as needed. In addition, forms such as batch culture, semi-batch culture, and continuous culture can be freely selected.
The concentration of TPA in the culture can be measured by high performance liquid chromatography, gas chromatography, gas chromatography mass spectrometer (GC-MS), liquid chromatography mass spectrometer (LC-MS) or the like.
Confirmation of the expression of the PAL and AbcDA genes can be performed by methods well known to those skilled in the art, such as Northern blotting, real-time PCR, and immunoblotting.
General molecular biology (Ausubel et al, Current Protocol in Molecular Biology
(2003); Sambrook et al, Molecular Cloning: a ^{A Laboratory Manual, 3 rd Edition (} 2001) , etc.), E. coli strain JM109 / pPAL-AbcDA can be improved, it is possible to improve the productivity of TPA. For example, for enzymes that are rate-limiting reactions in the TPA production pathway, increasing gene copy number, codon optimization, or amino acid substitution near the enzyme active site can increase enzyme activity and improve TPA productivity. it can. Moreover, productivity of TPA can be improved by destroying a gene of an enzyme that catalyzes a reaction for producing a by-product. Moreover, the method of repeatedly selecting the microbial cell which improved the TPA productivity by performing the mutation process by NTG or an ultraviolet-ray can also be utilized.

4). Example 2 for creating a microorganism that synthesizes terephthalic acid by a bio-method (R5-R6-R4)
E. coli strains capable of producing terephthalic acid (hereinafter referred to as TPA) are commonly used in molecular biology (Ausubel et al, Current Protocol in Molecular Biology (2003); Sambrook et al, Molecular Cloning: A Laboratory Manual, 3 ^rd Edition (2001) etc.) can be used in the following procedure.
Amycolatopsis orientalis-derived HMS gene (GenBank accession number AJ223998.1), Pseudomonas putida-derived mdlBCD gene (GenBank accession number: AY143338.1), Closrida bacterium-derived AbcDA gene (GenBank accession number: GU357991.1 GU357992.1) Thus, a plasmid pHMS-mdlBCD-AbcDA is constructed in which this ligated gene is incorporated into the downstream of the lac promoter of the Escherichia coli plasmid vector pHSG299 (manufactured by Takara Bio Inc.). If it is difficult to incorporate three types of DNA sequences into one vector, one of the DNA fragments may be introduced into a plasmid compatible with the pUC-type plasmid, for example, pMV119. For example, it is possible to construct pMV-AbcDA by introducing only the AbcDA gene into pMV119, and construct pHMS-mdlBCD by introducing the remaining HMS gene and mdlBCD gene into pUC19. Furthermore, a recombinant Escherichia coli strain retaining HMS, mdlBCD, and AbcDA is prepared by transforming Escherichia coli JM109 strain with the prepared plasmid DNA, and this Escherichia coli strain is named JM109 / pHMS-mdlBCD-AbcDA.
JM109 / pHMS-mdlBCD-AbcDA is obtained by methods well known to those skilled in the art (edited by the Japanese Society for Biotechnology, Biotechnology Experiments (2002); Vogel H. et al, Fermentation and Biochemical Engineering Handbook, 2nd Ed. (2007), etc.) It can be cultured. For example, E. coli JM109 / pHMS was added to 100 ml of a minimal synthetic medium supplemented with 20 g / L glucose, 50 μg / ml kanamycin, 0.2 mM IPTG (isopropyl-β-D-thiogalactopyranoside), and 50 μg / ml ampicillin if necessary. By seeding with -mdlBCD-AbcDA and using a flask, it can be cultured at 37 ° C. for 24 hours or more. When culturing using a jar fermenter, the substrate feed rate, the number of stirring revolutions, the aeration rate, the pH, and the like can be controlled within an appropriate range. An anaerobic condition, a microaerobic condition, and an aerobic condition can be selected as needed. In addition, forms such as batch culture, semi-batch culture, and continuous culture can be freely selected.
The concentration of TPA in the culture can be measured by high performance liquid chromatography, gas chromatography, gas chromatography mass spectrometer (GC-MS), liquid chromatography mass spectrometer (LC-MS) or the like.
Confirmation of the expression of HMS, mdlBCD and AbcDA genes can be carried out by methods well known to those skilled in the art, such as Northern blotting, real-time PCR, and immunoblotting.
General molecular biology (Ausubel et al, Current Protocol in Molecular Biology
(2003); Sambrook et al, Molecular Cloning: a ^{A Laboratory Manual, 3 rd Edition (} 2001) , etc.), E. coli strain JM109 / pHMS-mdlBCD-AbcDA can be improved, to improve the productivity of TPA it can. For example, for enzymes that are rate-limiting reactions in the TPA production pathway, increasing the gene copy number, codon optimization, or amino acid substitution near the enzyme active site can increase enzyme activity and improve TPA productivity. . Moreover, productivity of TPA can be improved by destroying a gene of an enzyme that catalyzes a reaction for producing a by-product. Moreover, the method of repeatedly selecting the microbial cell which improved the TPA productivity by performing the mutation process by NTG or an ultraviolet-ray can also be utilized.

5. Specific example of synthesizing TPA by chemically converting p-toluic acid synthesized by microorganisms (R8-R9-R1-R2-chemical conversion)
E. coli strains capable of producing p-toluic acid can be obtained by general molecular biology (Ausubel
et al, Current Protocol in Molecular Biology (2003); Sambrook et al, Molecular Cloning: By using the ^{A Laboratory Manual, 3 rd Edition (} 2001) , etc.), can be prepared by the following procedure.
Pseudomonas sp.-derived Propao gene (GenBank accession number: AB167410.1), Rhodococcus erythropolis.-derived DNA sequence encoding amidase (GenBank accession number: EU022986.1) was linked to the Escherichia coli plasmid vector pHSG299 (manufactured by Takara Bio Inc.) The plasmid pOxy-Ami incorporated into the downstream side of the lac promoter is constructed. On the other hand, a plasmid in which the linked gene of Clostridium difficle-derived HpdABC operon (GenBank accession number NC013316.1) and Closrida bacterium-derived AbcDA gene (GenBank accession number: GU357991.1 GU357992.1) is incorporated into the E. coli plasmid vector pMW119 (Nippon Gene) pHsdABC-AbcDA is constructed. It is important to find optimum conditions for the combination of the gene and plasmid to be introduced in addition to this. Furthermore, E. coli strain JM109 / pOxy-Ami-HsdABC-AbcDA is prepared by transforming E. coli strain JM109 with these plasmid DNAs.
JM109 / pOxy-Ami-HsdABC-AbcDA is a method well known to those skilled in the art (edited by Japan Society for Biotechnology, Biotechnology Experiments (2002); Vogel H. et al, Fermentation and Biochemical Engineering Handbook, 2nd Ed. (2007), etc. ). For example, E. coli JM109 / pOxy-Ami- is added to 100 ml of a minimal synthetic medium supplemented with 20 g / L glucose, 50 μg / ml ampicillin, 50 μg / ml kanamycin, 0.2 mM IPTG (isopropyl-β-D-thiogalactopyranoside). By inoculating HsdABC-AbcDA and using a flask, it can be cultured at 37 ° C. for 24 hours or more. When culturing using a jar fermenter, the substrate feed rate, the number of stirring revolutions, the aeration rate, the pH, and the like can be controlled within an appropriate range. An anaerobic condition, a microaerobic condition, and an aerobic condition can be selected as needed. In addition, forms such as batch culture, semi-batch culture, and continuous culture can be freely selected.
The concentration of p-toluic acid in the culture solution can be measured by high performance liquid chromatography, gas chromatography, gas chromatography mass spectrometer (GC-MS), liquid chromatography mass spectrometer (LC-MS) or the like. it can.
Confirmation of the expression of each gene can be performed by methods well known to those skilled in the art, such as Northern blotting, real-time PCR, and immunoblotting.
General molecular biology (Ausubel et al, Current Protocol in Molecular Biology
(2003); Sambrook et al, Molecular Cloning: a ^{A Laboratory Manual, 3 rd Edition (} 2001) , etc.), E. coli strain JM109 / pOxy-Ami-HsdABC- AbcDA can be improved, the productivity of p- toluic acid Can be improved. For example, for enzymes that are rate-limiting reactions in the p-toluic acid production pathway, increasing the gene copy number, codon optimization, or amino acid substitution near the enzyme active site increases the enzyme activity, producing p-toluic acid Can improve sex. In addition, the productivity of p-toluic acid can be improved by destroying the gene of an enzyme that catalyzes a reaction for producing a by-product. Moreover, the method of repeatedly selecting the microbial cell which improved the productivity of p-toluic acid by performing the mutation process by NTG or an ultraviolet-ray can also be utilized.
For the conversion of p-toluic acid to TPA, for example, a method described in US Pat. No. 3,089,906 (1963) is known. A p-toluic acid ester obtained by allowing methanol or the like to coexist in the reaction system is synthesized, converted into dimethyl terephthalic acid by an oxidation reaction, and further purified to a high purity to obtain terephthalic acid.

1 Synthetic path creation device 2 CPU (Central Processing Unit)
3 Memory 4 Hard disk drive 5 Portable media drive 6 Input device 7 Display device 8 External interface 10 Operation unit 11 Compound database 12 Known reaction database 13 Partial structure database 14 Common structure database 15 UF database 16, 17 Difference database 18 Virtual reaction Database 20 Storage unit 21 Control unit 22 Display unit

Claims (8)

  A method for producing terephthalic acid, comprising: converting phenylalanine into trans-cinnamic acid; converting trans-cinnamic acid into benzoic acid; and converting benzoic acid into terephthalic acid.   A medium containing a carbon source with a terephthalic acid producing ability, an enzyme that converts phenylalanine into trans-cinnamic acid, an enzyme that converts trans-cinnamic acid into benzoic acid, and an enzyme that converts benzoic acid into terephthalic acid A method for producing terephthalic acid, characterized in that terephthalic acid is accumulated in a medium or cells after culturing in the medium, and terephthalic acid is collected from the medium or cells.   A method for producing terephthalic acid, comprising a step of converting phenylpyruvic acid into mandelic acid, a step of converting mandelic acid into benzoic acid, and a step of converting benzoic acid into terephthalic acid.   A medium containing a carbon source having a terephthalic acid producing ability, an enzyme that converts phenylpyruvic acid to mandelic acid, an enzyme that converts mandelic acid to benzoic acid, and an enzyme that converts benzoic acid to terephthalic acid A method for producing terephthalic acid, characterized in that terephthalic acid is accumulated in a medium or cells after culturing in the medium, and terephthalic acid is collected from the medium or cells.   Converting phenylalanine to phenylacetamide, converting phenylacetamide to phenylacetic acid, converting phenylacetic acid to toluene, converting toluene to p-toluic acid, and converting p-toluic acid to terephthalic acid A method for producing terephthalic acid, comprising a step.   An enzyme that has the ability to produce p-toluic acid and converts phenylalanine to phenylacetamide, an enzyme that converts phenylacetamide to phenylacetic acid, an enzyme that converts phenylacetic acid to toluene, and an enzyme that converts toluene to p-toluic acid A method for producing terephthalic acid, comprising culturing an active microorganism in a medium containing a carbon source to produce p-toluic acid, and converting the obtained p-toluic acid into terephthalic acid.   A method for producing p-toluic acid, comprising a step of converting phenylalanine into phenylacetamide, a step of converting phenylacetamide into phenylacetic acid, a step of converting phenylacetic acid into toluene, and a step of converting toluene into p-toluic acid.   An enzyme that has the ability to produce p-toluic acid and converts phenylalanine to phenylacetamide, an enzyme that converts phenylacetamide to phenylacetic acid, an enzyme that converts phenylacetic acid to toluene, and an enzyme that converts toluene to p-toluic acid A method for producing p-toluic acid, comprising culturing an active microorganism in a medium containing a carbon source to produce p-toluic acid.

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