Method for producing aromatic carboxylic acids

Abstract

This invention provides a novel method for producing aromatic carboxylic acids from aromatic aldehydes. [Solution] A method for producing aromatic carboxylic acids, comprising reacting an aromatic aldehyde with an aldehyde dehydrogenase having 90% or more identity with an amino acid sequence selected from a group consisting of 20 specific amino acid sequences, each with a different sequence. The method may include transforming a microorganism with a gene encoding the aldehyde dehydrogenase. The aromatic aldehyde may be furfural or its substituted derivatives.

Description

Technical Field

[0001] Techniques related to the production of aromatic carboxylic acids are disclosed.

Background Art

[0002] Aromatic carboxylic acids are a group of chemical substances that play important roles in fields such as the chemical industry and the pharmaceutical and agrochemical industries. For example, 2,5-furandicarboxylic acid (FDCA) is an aromatic dicarboxylic acid that can be synthesized from biomass, and has attracted attention in recent years as a monomer raw material for bio-based polymers such as polyethylene furanoate (PEF). Aromatic carboxylic acids are generally produced by oxidation at the benzyl position, starting from a compound in which the carboxy group thereof is substituted with an alkyl group, a hydroxymethyl group, a formyl group, or the like. Regarding the aforementioned FDCA, it can be synthesized by 6-electron oxidation of 5-(hydroxymethyl)furfural (HMF) obtained by dehydration of biomass-derived hexose monosaccharides.

[0003] Bioconversion processes using enzymes, microbial cells, or the like as catalysts can carry out oxidation reactions under mild conditions, and are therefore useful as methods for producing aromatic carboxylic acids. In the production of bulk chemicals, it is most preferable to use oxygen as an oxidizing agent. By using an oxidase that can directly utilize oxygen as an electron acceptor, or by coupling dehydrogenase and respiration in living microbial cells, a green reaction using oxygen as an oxidizing agent in an aqueous solvent at normal temperature can be realized.

[0004] For example, Non-Patent Documents 1 and 2 disclose methods for producing FDCA by allowing an oxidase called HMF oxidase (HMFO) to act on HMF. According to this method, it is possible to perform the conversion of HMF to FDCA using only one type of enzyme and oxygen as an oxidizing agent. However, since HMFO used in this method has low reactivity with the intermediate 5-formyl-2-furancarboxylic acid (FFA), there is a problem that the reaction does not complete and FFA accumulates.

[0005] Patent Document 1 discloses a method for oxidative conversion of HMF to FDCA using Pseudomonas putida into which genes for HMF oxidase, transporter, and aldehyde dehydrogenase have been introduced. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Special Publication 2014-508531 [Non-patent literature]

[0007] [Non-Patent Document 1] Willem P. Dijkman, Marco W. Fraaije. Applied and Environmental Microbiology. 2014, 80(3), 1082-1090 [Non-Patent Document 2] Willem P. Dijkman, Claudia Binda, Marco W. Fraaije, and Andrea Mattevi. ACS Catalysis. 2015, 5, 1833-1839 [Overview of the project] [Problems that the invention aims to solve]

[0008] One challenge is to provide a new method for producing aromatic carboxylic acids from aromatic aldehydes. [Means for solving the problem]

[0009] The inventions described below are provided. Item 1 A method for producing aromatic carboxylic acids, comprising reacting an aromatic aldehyde with an aldehyde dehydrogenase having 90% or more identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20. Section 2 A method for producing an aromatic carboxylic acid, comprising causing a microorganism that expresses an aldehyde dehydrogenase having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20 to act on an aromatic aldehyde. Claim 3 The method according to Claim 2, further comprising transforming a microorganism with a gene encoding an aldehyde dehydrogenase having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20. Claim 4 The method according to Claim 2 or 3, wherein the microorganism is a Gram-negative bacterium. Claim 5 The method according to Claim 4, wherein the Gram-negative bacterium is Escherichia coli. Claim 6 The gene is under the control of a lactose-inducible promoter, comprising the step of culturing the transformed microorganism in a medium containing lactose and glucose, The method according to Claim 3. Claim 7 The method according to any one of Claims 1 to 6, wherein the aromatic aldehyde is furfural or a substituted product thereof. Claim 8 The method according to Claim 7, wherein the aromatic aldehyde is furfural. Claim 9 The method according to Claim 7, wherein the aromatic aldehyde is hydroxymethylfurfural. Claim 10 The method according to Claim 7, wherein the aromatic aldehyde is 2,5-diformylfuran. Claim 11 The method according to Claim 7, wherein the aromatic aldehyde is 5-formyl-2-furancarboxylic acid. Effects of the Invention

[0010] A means for producing an aromatic carboxylic acid from an aromatic aldehyde is provided. Brief Description of the Drawings

[0011] [Figure 1]This is the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter calcoaceticus. [Figure 2] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter kyonggiensis. [Figure 3] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter gyllenbergii. [Figure 4] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter soli. [Figure 5] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter sichuanensis. [Figure 6] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter pittii. [Figure 7] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Pseudomonas duriflava. [Figure 8] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Stutzerimonas stutzeri. [Figure 9] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Pseudomonas putida. [Figure 10] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Halomonas nigrificans. [Figure 11] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Pseudomonas nitroreducens. [Figure 12] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Pseudomonas putida. [Figure 13] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Stenotrophomonas maltophilia. [Figure 14] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Stenotrophomonas rhizophila. [Figure 15] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Paraburkholderia caballeronis. [Figure 16] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Paraburkholderia bryophila. [Figure 17] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Halotalea alkalilenta. [Figure 18] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Marinomonas polaris. [Figure 19] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Stenotrophomonas maltophilia. [Figure 20] This is the amino acid sequence of benzaldehyde dehydrogenase derived from Burkholderia cepacia. [Figure 21] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter chalcaceticus. [Figure 22] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter kyonggiensis. [Figure 23] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter gyllenbergii. [Figure 24] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter soli. [Figure 25] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter sichuanensis. [Figure 26] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter pittii. [Figure 27] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Pseudomonas duriflava. [Figure 28] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Stutzerimonas stutzeri. [Figure 29] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Pseudomonas putida. [Figure 30] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Halomonas nigrificans. [Figure 31] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Pseudomonas nitroreducens. [Figure 32] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Pseudomonas putida. [Figure 33] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Stenotrophomonas maltophilia. [Figure 34] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Stenotrophomonas rhizophila. [Figure 35] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Paraburkholderia caballeronis. [Figure 36] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Paraburkholderia bryophila. [Figure 37] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Halotalea alkalilenta. [Figure 38] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Marinomonas polaris. [Figure 39] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Stenotrophomonas maltophilia. [Figure 40] This is the base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase derived from Burkholderia cepacia. [Figure 41] The results of measuring the amount of furfural in Example 1 are shown. "IPTG-EV" refers to the case where E. coli transformed with an empty vector and IPTG was induced, "IPTG EX" refers to the case where E. coli transformed with the gene encoding AcBADH and IPTG was induced, "Auto EV" refers to the case where E. coli transformed with an empty vector and autoinduction was performed, and "Auto EX" refers to the case where E. coli transformed with the gene encoding AcBADH and autoinduction was performed. [Figure 42] The results of measuring the amount of 2-furoic acid in Example 1 are shown. "IPTG-EV" refers to the case where E. coli transformed with an empty vector and IPTG was induced, "IPTG EX" refers to the case where E. coli transformed with the gene encoding AcBADH and IPTG was induced, "Auto EV" refers to the case where E. coli transformed with an empty vector and autoinduction was performed, and "Auto EX" refers to the case where E. coli transformed with the gene encoding AcBADH and autoinduction was performed. [Figure 43] The results of measuring the DO and pH of the culture medium in Example 2 are shown below. [Figure 44] In Example 2, the cumulative amount of MHF input and the conversion rate from HMF to HMFCA were determined, and the results are shown. [Figure 45] The results of measuring the amounts of FFA and FDCA in Example 3 are shown below. [Modes for carrying out the invention]

[0012] The aldehyde dehydrogenase preferably has a certain degree of identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20. SEQ ID NO: 1 is the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter calcoaceticus. Acinetobacter calcoaceticus is also known as Acinetobacter guillouiae. SEQ ID NO: 2 is the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter kyonggiensis. SEQ ID NO: 3 is the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter gyllenbergii. SEQ ID NO: 4 is the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter soli. SEQ ID NO: 5 is the amino acid sequence of benzaldehyde dehydrogenase derived from Acinetobacter sichuanensis. Sequence ID 6 is the amino acid sequence of benzaldehyde dehydrogenase from Acinetobacter pittii. Sequence ID 7 is the amino acid sequence of benzaldehyde dehydrogenase from Pseudomonas duriflava. Sequence ID 8 is the amino acid sequence of benzaldehyde dehydrogenase from Stutzerimonas stutzeri. Sequence ID 9 is the amino acid sequence of benzaldehyde dehydrogenase from Pseudomonas putida. Sequence ID 10 is the amino acid sequence of benzaldehyde dehydrogenase from Halomonas nigrificans. Sequence ID 11 is the amino acid sequence of benzaldehyde dehydrogenase from Pseudomonas nitroreducens. Sequence ID 12 is the amino acid sequence of benzaldehyde dehydrogenase from Pseudomonas putida. Pseudomonas putida is also known as Pseudomonas bharatica. Sequence ID 13 is the amino acid sequence of benzaldehyde dehydrogenase from Stenotrophomonas maltophilia.Sequence ID 14 is the amino acid sequence of benzaldehyde dehydrogenase from Stenotrophomonas rhizophila. Sequence ID 15 is the amino acid sequence of benzaldehyde dehydrogenase from Paraburkholderia caballeronis. Sequence ID 16 is the amino acid sequence of benzaldehyde dehydrogenase from Paraburkholderia bryophila. Sequence ID 17 is the amino acid sequence of benzaldehyde dehydrogenase from Halotalea alkalilenta. Sequence ID 18 is the amino acid sequence of benzaldehyde dehydrogenase from Marinomonas polaris. Sequence ID 19 is the amino acid sequence of benzaldehyde dehydrogenase from Stenotrophomonas maltophilia. Sequence ID 20 is the amino acid sequence of benzaldehyde dehydrogenase from Burkholderia cepacia.

[0013] "Above a certain level" means, for example, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In one embodiment, the aldehyde dehydrogenase preferably has a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 12.

[0014] The identity of amino acid sequences can be evaluated by any means known in the field. For example, it can be calculated using commercially available analytical tools or tools available via telecommunication lines (the Internet). As an example, the identity of amino acid sequences can be calculated by using the default parameters of the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / .

[0015] In one embodiment, if the amino acid sequence of the aldehyde dehydrogenase has an amino acid sequence in which one or several amino acids are substituted for the amino acid sequence of SEQ ID NO: 1, the type of amino acid substitution is not particularly limited, but it is preferable that it be a conservative amino acid substitution. Examples of conservative substitutions include, but are not limited to, substitutions between basic amino acids (H, K, R), substitutions between acidic amino acids (D, E), substitutions between neutral nonpolar amino acids (A, V, L, I, P, F, M, W), substitutions between neutral polar amino acids (G, N, Q, S, T, V, C), substitutions between aromatic amino acids (W, F, H, Y), substitutions between nitrogen-containing amino acids (K, R, N, Q, P), substitutions between sulfur-containing amino acids (C, M), substitutions between oxygen-containing amino acids (S, T), substitutions between β-branched-chain amino acids (V, L, I), and substitutions between amino acids (A, G) having linear alkyl or hydrogen side chains.

[0016] Aromatic aldehydes are compounds in which an aldehyde group is bonded to an aromatic compound (aromatic ring), and their type is not particularly limited. Examples of aromatic compounds include benzene, furan, thiophene, pyrrole, pyrazole, imidazole, pyridine, pyridazine, pyrimidine, pyrazine, benzofuran, naphthalene, quinoline, isoquinoline, and azulene. The number of aldehyde groups bonded to the aromatic compound is not particularly limited, but for example, it may be one, two, or three, and preferably one or two.

[0017] Specific examples of aromatic aldehydes include, for example, benzaldehyde, methylbenzaldehyde, hydroxybenzaldehyde, methoxybenzaldehyde, chlorobenzaldehyde, nitrobenzaldehyde, 3,4-methylenedioxybenzaldehyde, 2,3-methylenedioxybenzaldehyde, cinnamaldehyde, vanillin, p-tolualdehyde, 4-(hydroxymethyl)benzaldehyde, terephthalaldehyde, terephthalaldehyde acid, furfural, hydroxymethylfurfural, 2,5-diformylfuran, 5-formyl-2-furancarboxylic acid, 5-methyl-2-flualdehyde, 2-pyridinecarboxyaldehyde, 3-pyridinecarboxyaldehyde, and 4-pyridinecarboxyaldehyde.

[0018] In one embodiment, preferred aromatic aldehydes are furfural, hydroxymethylfurfural, 2,5-diformylfuran, 5-formyl-2-furancarboxylic acid, or 5-methyl-2-flualdehyde.

[0019] Aromatic aldehydes may be added directly to the reaction system, but they can also be supplied to the reaction system by adding them as a precursor, such as an aromatic alcohol, and then producing the aromatic aldehyde from the precursor using another suitable enzyme. For example, enzymes that produce aromatic aldehydes from aromatic alcohols include alcohol dehydrogenase, alcohol oxidase, peroxidase, and laccase.

[0020] The aldehyde dehydrogenase preferably has the activity to catalyze the dehydrogenation of the above aromatic aldehyde. Here, the aromatic aldehyde is preferably one or more, two or more, three or more, four or more, or five types selected from the group consisting of furfural, hydroxymethylfurfural, 2,5-diformylfuran, 5-formyl-2-furancarboxylic acid, and 5-methyl-2-furaldehyde.

[0021] The method for reacting aldehyde dehydrogenase with aromatic aldehydes is arbitrary. For example, the reaction can be carried out by contacting the aromatic aldehyde with aldehyde dehydrogenase under conditions suitable for the aldehyde dehydrogenase to exhibit its activity in catalyzing the dehydrogenation of the aromatic aldehyde. For example, by contacting aldehyde dehydrogenase with aromatic aldehyde in a suitable buffer solution, the aromatic aldehyde can be dehydrogenated (oxidized) to produce an aromatic carboxylic acid. Here, an aromatic carboxylic acid is obtained by changing the aldehyde group of an aromatic aldehyde to a carboxylic acid.

[0022] In one embodiment, the aromatic carboxylic acid is preferably 2-furoic acid, 5-(hydroxymethyl)-2-furanic acid, 5-formyl-2-furanic acid, or 2,5-franic acid.

[0023] The buffer is not particularly limited, but examples include Bicine, Glycine, MES, Bis-Tris, Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, and Na2HPO4 buffer.

[0024] The reaction conditions for reacting an aromatic aldehyde with aldehyde dehydrogenase are not particularly limited and can be appropriately set depending on the characteristics of the aldehyde dehydrogenase, the type of reagent used, etc. The reaction temperature can be set, for example, in the range of 10°C to 90°C. In one embodiment, the reaction temperature is preferably 20°C or higher, 25°C or higher, 30°C or higher, 35°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, or 65°C or higher, and preferably 90°C or lower, 85°C or lower, 80°C or lower, or 75°C or lower. These lower and upper temperature limits can be combined arbitrarily.

[0025] The pH during the reaction can be appropriately set within the range of, for example, 4 to 12. In one embodiment, the pH during the reaction can be set to 5 or higher, 6 or higher, 7 or higher, or 8 or higher, and 11 or lower, or 10 or lower. These lower and upper limits of pH can be appropriately combined to form a range. In one embodiment, the pH during the reaction is preferably 8.5 or higher and 11 or lower.

[0026] The reaction time can be set appropriately depending on the purpose. For example, the reaction time can be set within a range of 1 hour to 1 week. In one embodiment, the reaction time can be 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 3 days or more, 4 days or more, 5 days or more, or 6 days or more, and 7 days or less, 6 days or less, 5 days or less, 4 days or less, or 3 days or less. These lower and upper limits of the reaction time can be combined as appropriate to form a range.

[0027] The concentration or amount of aldehyde dehydrogenase used can be appropriately set depending on the purpose. For example, the concentration of polypeptide in the solvent can be appropriately set within the range of 10 nM to 5000 nM. In one embodiment, the concentration of aldehyde dehydrogenase in the solvent during the reaction is preferably 50 nM to 200 nM, 75 nM to 150 nM, or 80 nM to 130 nM.

[0028] Aldehyde dehydrogenase may be used as an isolated or purified enzyme, but it can also be used in the form of a microorganism that expresses it. Such a microorganism can be obtained, for example, by introducing a gene encoding aldehyde dehydrogenase into the microorganism and causing the microorganism to express the gene.

[0029] The gene encoding aldehyde dehydrogenase is arbitrary, but preferably a polynucleotide having a certain degree of identity with a base sequence selected from the group consisting of, for example, SEQ ID NOs: 21 to 40 (preferably the base sequence of SEQ ID NOs: 21 or 32). A certain degree of identity means, for example, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The polynucleotide may be DNA or RNA.

[0030] The identity of base sequences, like that of amino acid sequences, can be calculated using commercially available analysis tools or tools accessible via telecommunication lines (the internet). For example, software such as FASTA, BLAST, PSI-BLAST, and SSEARCH can be used for calculation. Specifically, the main initial conditions commonly used for BLAST searches are as follows. That is, in Advanced BLAST 2.1, the identity (%) of nucleotide sequences can be calculated by using the blastn program and setting various parameters to their default values.

[0031] Polynucleotides can be easily obtained using chemical DNA synthesis methods (e.g., phosphoamidite method) or genetic engineering techniques based on a base sequence selected from a group consisting of 21 to 40 bases. Sequence ID No. 21 is a base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Acinetobacter calcoaceticus. Acinetobacter calcoaceticus is also known as Acinetobacter guillouiae. Sequence ID No. 22 is a base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Acinetobacter kyonggiensis. Sequence ID No. 23 is a base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Acinetobacter gyllenbergii. Sequence ID No. 24 is a base sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Acinetobacter soli. Sequence ID 25 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Acinetobacter sichuanensis. Sequence ID 26 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Acinetobacter pittii. Sequence ID 27 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Pseudomonas duriflava. Sequence ID 28 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Stutzerimonas stutzeri. Sequence ID 29 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Pseudomonas putida. Sequence ID 30 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Halomonas nigrificans. Sequence ID 31 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Pseudomonas nitroreducens.Sequence ID 32 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Pseudomonas putida. Sequence ID 33 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Stenotrophomonas maltophilia. Sequence ID 34 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Stenotrophomonas rhizophila. Sequence ID 35 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Paraburkholderia caballeronis. Sequence ID 36 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Paraburkholderia bryophila. Sequence ID 37 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Halotalea alkalilenta. Sequence ID 38 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Marinomonas polaris. Sequence ID 39 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Stenotrophomonas maltophilia. Sequence ID 40 is a nucleotide sequence encoding the amino acid sequence of benzaldehyde dehydrogenase from Burkholderia cepacia.

[0032] In one embodiment, it is preferable that the gene encoding aldehyde dehydrogenase is introduced into the microorganism (host) in a form that allows for expression, incorporated into the vector. The type of vector can be appropriately selected considering the purpose of use and the type of host cell. For example, a vector selected from plasmid vectors, cosmid vectors, phage vectors, viral vectors (adenovirus vectors, retrovirus vectors, herpesvirus vectors, etc.) can be used.

[0033] The gene encoding the aldehyde dehydrogenase incorporated into the vector can be placed under the control of any promoter. In one embodiment, the gene is preferably placed under the control of a lactose-inducible promoter.

[0034] The type of microorganism into which the gene is introduced is not particularly limited, as long as it can express aldehyde dehydrogenase. In the microorganism (host cell), the gene or vector may exist autonomously or be incorporated into the genome homologously or non-homologously. The microorganism may be a prokaryotic cell or a eukaryotic cell. In one embodiment, the microorganism is preferably a Gram-negative bacterium, and more preferably Escherichia coli. Other suitable microorganisms include yeast and filamentous fungi.

[0035] By culturing a microorganism into which a gene encoding aldehyde dehydrogenase has been introduced under conditions suitable for the invention of the gene, it is possible to obtain a microorganism that expresses the gene and produces aldehyde dehydrogenase. The culture conditions are arbitrary, and when an inducible promoter is used for gene expression, it is preferable to add a promoter inducer to the culture medium before culturing. Examples of promoter inducers include IPTG and lactose.

[0036] When aldehyde dehydrogenase is used in the form of a microorganism that produces it, aromatic carboxylic acids can be produced by allowing the microorganism to act on aromatic aldehydes while being cultured. Culture conditions can be appropriately set depending on the type of microorganism, for example. Typically, culture can be performed at 10-42°C, preferably 18-37°C, with a pH in the range of 4-12, for 5-168 hours, preferably 8-72 hours. Depending on the host, either shaking culture or static culture is possible, but stirring and / or aeration may be performed as needed.

[0037] The electrons of NADH produced by the dehydrogenation of aromatic aldehydes are thought to be ultimately donated to oxygen via the respiratory chain electron transport system and other pathways. Therefore, maintaining the cellular respiratory activity is desirable for the reaction to proceed. For this reason, in one embodiment, it is preferable not to subject the cells to excessive stress by overexpressing the protein, and it is preferable to optimize the protein expression intensity by appropriately selecting the promoter and induction conditions.

[0038] When using enzyme-expressing cells as a catalyst, oxygen is consumed as the terminal oxidizing agent, so it is preferable to proceed with the reaction while aerating and stirring. Examples of gases supplied to the reaction system include air or pure oxygen. Pressurization can be applied as needed.

[0039] Aromatic aldehydes are generally cytotoxic, so it is preferable to add them in small amounts. They may be added in batches in small amounts, or continuously using a pump or the like. When adding in batches, it is preferable to add them when the previously added raw materials have been consumed. As long as the raw materials remain, DO consumption and pH decrease will be observed due to the reaction, so the timing of addition can be determined by the increase in DO and the cessation of the pH decrease.

[0040] Aldehyde dehydrogenase can be isolated and used from the microorganism that produces it or from its culture supernatant. Purification or isolation of aldehyde dehydrogenase from the culture supernatant can be carried out by appropriately combining known methods. For example, methods include ammonium sulfate precipitation, solvent precipitation such as ethanol, dialysis, ultrafiltration, acid extraction, and various chromatography techniques (e.g., gel filtration chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, high-performance liquid chromatography, etc.). As a support for affinity chromatography, for example, a support to which an antibody against the polypeptide is conjugated, or, if a peptide tag is attached to the polypeptide, a support to which a substance with affinity for this peptide tag is conjugated can be used.

[0041] If aldehyde dehydrogenase accumulates within the cells of microorganisms, the microorganisms can be lysed, and the polypeptide can be purified or isolated from the supernatant after centrifugation of the lysate in the same manner as described above. For example, after the culture is complete, the cells collected by centrifugation are suspended in a cell lysate buffer (20-100 mM Tris-HCl (pH 8.0), 5 mM EDTA), sonicated, and the lysate is centrifuged at 10,000-15,000 rpm for 10-15 minutes to obtain the supernatant. The precipitate after centrifugation can be further purified after solubilization with guanidium hydrochloride or urea as needed. In this case, it is preferable to add a coenzyme (e.g., NAD) to the reaction system. Coenzymes can be used in quantifiable amounts, but the amount added can be made catalytic by coexisting with a suitable coenzyme regeneration system.

[0042] As aromatic carboxylic acids are produced, the pH of the reaction system decreases. Therefore, it is preferable to maintain the pH of the reaction solution within a suitable range by adding a neutralizing agent. Sodium hydroxide, calcium carbonate, ammonia, etc., can be used as neutralizing agents.

[0043] The aromatic carboxylic acids produced by the above method can be recovered and purified from the reaction solution or culture medium. Recovery and purification can be carried out by appropriately combining known methods. For example, methods using filtration, centrifugation, distillation, crystallization, organic solvent extraction, activated carbon adsorption, ion exchange chromatography, normal-phase chromatography, or reverse-phase chromatography can be used. These operations may also be combined with chemical treatments (e.g., esterification with alcohol). [Examples]

[0044] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0045] 1. Production of 2-furonic acid from furfural using enzyme-expressing microorganisms <Enzyme selection and expression vector construction> Referencing entry number RC00075 in the KEGG database, we obtained the EC numbers for a series of enzymes known to be responsible for the oxidation of aromatic aldehydes. Referencing these EC numbers in the BRENDA database, we selected benzaldehyde dehydrogenase (EC. 1.2.1.28) from Acinetobacter chalcaceticus as an enzyme with relatively high specific activity and turnover rate. The DNA sequence encoding this enzyme protein (NCBI Accession: AAC32670.1) was designed using a web tool (https: / / www.vectorbuilder.jp / tool / codon-optimization.html) to match the codon frequency of E. coli K-12 strain, and artificial gene synthesis (Genscript) was performed. The synthesized DNA was incorporated between the NdeI-XhoI sites of the E. coli expression vector pET-28a(+) to construct the expression vector pET28a-AcBADH. This expression vector expresses an enzyme protein with a 6×His tag at the N-terminus.

[0046] <Culture of enzyme-expressing E. coli> pET28a-AcBADH was added to ECOS competent E. coli BL21(DE3) (Nippon Gene), and transformation was performed according to the manufacturer's recommended protocol. Transformants were selected on LB+Kan agar. As a negative control, a blank vector (pET-28a(+)) was used for transformation in the same manner. Several colonies from the agar were inoculated into 5 mL of LB+Kan liquid medium containing 0.2% glucose and incubated overnight at 37°C and 180 rpm (amplitude 20 mm, reciprocating). Next, this was used as a seed culture medium for expression induction by IPTG and autoinduction. For IPTG induction, 200 μL of pre-culture solution was inoculated into 20 mL of LB+Kan liquid medium in a 300 mL baffled Erlenmeyer flask. The culture was incubated at 37°C and 250 rpm (amplitude 20 mm, swirling) until OD660 = 0.4-0.6. After incubation, IPTG was added to a final concentration of 0.4 mM, and expression was achieved by incubation overnight at 18°C ​​and 250 rpm. For autoinduction, 1 mL of pre-culture solution was inoculated into 20 mL of autoinduction medium (10 g / L Tryptone, 5 g / L Yeast extract, 10 g / L NaCl, 12.5 g / L K2HPO4, 2.3 g / L KH2PO4, 0.5 g / L glucose, 2 g / L lactose, 50 mg / L kanamycin sulfate) in a 300 mL baffled Erlenmeyer flask. The culture was incubated at 18°C ​​and 250 rpm for 24 hours with shaking.

[0047] <Reaction> E. coli expressing enzymes by IPTG induction and autoinduction were recovered by centrifugation and suspended separately in 100 mM Tris-HCl (pH 7.5) to achieve an OD660 of 4. 5 mL of these suspensions were transferred to a 100 mL baffled Erlenmeyer flask, and 20 mM furfural (Tokyo Chemical Co., Ltd.) was added. The reaction was initiated by shaking at 30°C and 250 rpm (amplitude 25 mm, swirling). Analysis of the furfural and furic acid in the supernatant after 1.25 hours showed that furfural had disappeared. Therefore, 80 mM furfural and 20 g / L (200 mM) calcium carbonate as a neutralizing agent were added 2 hours after the start of the reaction, and the reaction was continued. Analysis of furfural and furic acid in the reaction solution was performed at 4 hours and 22 hours after the start of the reaction. The results are shown in Figures 41 and 42. When cells induced by IPTG were used as a catalyst, 33 mM furfural acid was produced at 22 hours of reaction, while 86 mM furfural acid was produced when cells induced by autoinduction were used. The residual furfural concentration was 47 mM when expression was induced by IPTG and 0.6 mM when induced by autoinduction. When transformants using empty vectors were used, almost no 2-furfural acid was produced regardless of the induction conditions.

[0048] <Analysis of furfural and 2-furic acid> The reaction mixture was centrifuged, and the supernatant was measured for ultraviolet absorption at two wavelengths (277.5 nm and 245 nm) using a micro-spectrophotometer to quantify furfural and 2-furonic acid. Furfural and 2-furonic acid showed maximum absorption at 277.5 nm and 245 nm, respectively. The absorption at 277.5 nm was observed only by furfural, while the absorption at 245 nm was influenced by both furfural and 2-furonic acid. Since the concentrations of the substrate and product were sufficiently high that the influence of ultraviolet absorption by interfering components could be ignored, A277.5 and A245 can be expressed by the following two equations. A277.5 = ε1d C1 A245 = ε2d C1 + ε3d C2 Solving these for C1 and C2 yields the following result. C1 = A277.5 / ε1d C2 = (A245 - ε2 A277.5 / ε1) / ε3d Here, C1 is the molar concentration of furfural, C2 is the molar concentration of 2-furic acid, ε1 is the extinction coefficient of furfural at 277.5 nm, ε2 is the extinction coefficient of furfural at 245 nm, and ε3 is the extinction coefficient of 2-furic acid at 245 nm. By pre-measuring the absorbance of furfural and furic acid standards, ε1, ε2, and ε3 were obtained to be 14.6 Abs mM each. -1 cm -1 , 2.56 Abs mM -1 cm -1 , 10.7 Abs mM -1 cm -1 That was the decision.

[0049] 2. Production of HMFCA from HMF using enzyme-expressing microorganisms (scale scaling) <Preculture> Approximately 10 colonies of E. coli BL21(DE3) / pET28a-AcBADH were scraped from agar plates using a loop, inoculated into 50 mL of LB+Kan liquid medium in a 500 mL Sakaguchi flask, and incubated overnight at 37°C with shaking at 115 rpm (amplitude 70 mm). Two flasks were used for incubation, and the combined culture medium was used as the seed culture for the main culture.

[0050] <Main culture> After inoculating 75 mL of pre-culture solution into 1.5 L of auto-induction medium (containing 0.2 g / L adecanol LG-126) loaded into a 3 L jar fermenter (ABLE), the culture was incubated at 18°C, with an aeration rate of 0.75 L / min and a stirring speed of 350 rpm (total oxygen transfer capacity coefficient kLa = 100 h). -1 The cells were cultured for 27 hours under a pressure of 0.05 MPa to obtain a culture solution of enzyme-expressing bacteria (OD660 = 6.4).

[0051] <Reaction> The culture solution in the jar fermenter was heated to 30°C, with an aeration rate of 0.75 L / min and a stirring speed of 500 rpm (kLa=250 h -1 ), and aerated stirring was performed at atmospheric pressure. As the initial raw material, 3.87 g (30.7 mmol) of HMF (Angene International Limited) was added to the culture solution to start the reaction. The pH decrease and DO consumption accompanying the reaction were monitored, and 3.87 g of HMF and 6 mL of 5 M NaOH were added to the culture solution when an increase in DO due to HMF depletion was observed. The addition of HMF and NaOH was performed a total of 12 times, and a cumulative total of 46.4 g (369 mmol) of HMF was charged into the reaction system. The reaction was carried out for 5.8 hours, the sampled culture solution was centrifuged, and HMF and HMFCA in the supernatant were analyzed by HPLC. No HMF was detected at the end of the reaction, and 1.59 L of a reaction solution containing 32.1 g / L (226 mM) of HMFCA was obtained (reaction yield 97.5%). The results of measuring DO and pH of the culture solution are shown in Figure 43, and the results of determining the cumulative input amount of MHF and the conversion rate from HMF to HMFCA are shown in Figure 44.

[0052] <HPLC analysis> Analysis of HMF, HMFCA and the like by HPLC was carried out as follows. HPLC apparatus: Chromaster (Hitachi, Ltd.) Column: COSMOSIL 5C18 PAQ (4.6 mm I.D. × 250 mm) (Nacalai Tesque) Mobile phase: 20 mM sodium phosphate buffer (pH 7.0):methanol = 80:20 (volume ratio) Flow rate: 1 mL / min Column temperature: 40°C Detection: UV absorption (260 nm) Injection volume: 10 μL Commercial reagents for HMF, DFF, HMFCA, FFA, and FDCA (all from Tokyo Chemical Industry Co., Ltd.) were dissolved in 100 mM NaPB (pH 7.0) to a concentration of 2 mM each, and diluted with the mobile phase to form standard samples. A dilution series from 10 μM to 200 μM was analyzed, and the compounds were quantified based on a calibration curve created using the peak area.

[0053] 3. Production of FDCA from FFA using enzymes <expression> 1 mL of pre-culture solution of E. coli BL21(DE3) / pET28a-AcBADH was inoculated into 50 mL of LB+Kan liquid medium in a 500 mL Sakaguchi flask, and cultured with shaking at 37°C and 115 rpm (amplitude 70 mm) for several hours. When the OD660 reached 0.4-0.6, 0.4 mM IPTG was added, and the cells were cultured overnight with shaking at 18°C ​​and 115 rpm to express the target protein. After the culture was completed, the cells were collected from the entire culture by centrifugation, and lysate was obtained by adding 5 mL of Bugbuster master mix (Merck) and gently shaking at room temperature. After removing insoluble matter by centrifugation, the crude enzyme solution was prepared by passing it through a microfiltration membrane (Kurabo) with a pore size of 0.2 μm.

[0054] <Enzyme Purification> Enzyme proteins were purified by immobilized metal affinity chromatography. The entire crude enzyme solution was applied to a 1 mL TALON® column (Cytiva) pre-equilibrated with binding buffer (100 mM potassium phosphate buffer (KPB), 300 mM NaCl, pH 7.5). After removing contaminating proteins by passing 10 mL of binding buffer through the column, the target protein was eluted by passing 5 mL of elution buffer (100 mM KPB, 300 mM NaCl, 150 mM imidazole, pH 7.5) through the column. The eluted sample was buffer-exchanged with the final buffer (50 mM KPB, pH 7.0) using an Amicon Ultra 15 30kDa (Merck) to obtain the purified sample. The weight concentration of the enzyme in the sample was calculated using the absorbance at 280 nm and the absorption coefficient of the protein calculated from the amino acid sequence. ProtParam (https: / / web.expasy.org / protparam / ) was used to calculate the extinction coefficient based on the amino acid sequence.

[0055] <Reaction> A purified enzyme was added to 10 mM FFA, 10 mM NAD+, and 100 mM NaPB (pH 7.0) at a final concentration of 0.2 g / L (4 μM), and the reaction was carried out under static conditions at 30°C for 6 hours. Samples were taken every 2 hours from the start of the reaction, and the substrate and product were analyzed by TLC. A decrease in FFA and the formation of FDCA were observed during the reaction, and the conversion rate reached 95% after 6 hours of reaction. The measurement results are shown in Figure 45.

[0056] <Analysis> A 1 μL sample was applied to silica gel 60 F254 (Merck), and after development using a mixed solvent of 1-butanol:acetic acid:water = 4:1:1 (volume ratio), spots were detected by irradiation with 254 nm UV. Similar to step 2 above, samples of known concentrations were prepared by dissolving five different compounds at concentrations of 1-20 mM in 100 mM NaPB (pH 7.0), and these were also analyzed simultaneously. After converting the TLC images to grayscale (32-bit) using ImageJ, the absorbance was calculated by dividing each pixel value by the background value, logarithmically transforming the value, and multiplying by -1. Spot intensity was quantified by integrating the absorbance over the entire spot, and the compounds were quantified using a calibration curve created from the spot intensities.

Claims

1. A method for producing aromatic carboxylic acids, comprising reacting an aromatic aldehyde with an aldehyde dehydrogenase having 90% or more identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20.

2. A method for producing aromatic carboxylic acids, comprising reacting an aromatic aldehyde with a microorganism that expresses an aldehyde dehydrogenase having 90% or more identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20.

3. The method according to claim 2, further comprising transforming a microorganism with a gene encoding an aldehyde dehydrogenase having 90% or more identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20.

4. The method according to claim 2 or 3, wherein the microorganism is a Gram-negative bacterium.

5. The method according to claim 4, wherein the Gram-negative bacterium is Escherichia coli.

6. The aforementioned gene is under the control of a lactose-inducible promoter, The process includes culturing the transformed microorganisms in a medium containing lactose and glucose. The method according to claim 3.

7. The method according to any one of claims 1 to 6, wherein the aromatic aldehyde is furfural or a substituted product thereof.

8. The method according to claim 7, wherein the aromatic aldehyde is furfural.

9. The method according to claim 7, wherein the aromatic aldehyde is hydroxymethylfurfural.

10. The method according to claim 7, wherein the aromatic aldehyde is 2,5-diformylfuran.

11. The method according to claim 7, wherein the aromatic aldehyde is 5-formyl-2-furancarboxylic acid.

Images