Recombinant microorganism, recombinant protein, and production method for c6 compound

Abstract

To provide a recombinant microorganism, a recombinant protein, and a production method enabling production of a C6 compound.SOLUTION: A recombinant microorganism comprises a heterologous gene encoding a protein having at least one reductive activity between the activity of reducing and transforming muconic acid to 2-hexenedioic acid and the activity of reducing and transforming 2-hexenedioic acid to adipic acid, where the heterologous gene has a specific amino acid sequence or comprises a specific polynucleotide sequence.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a recombinant microorganism, a recombinant protein, and a method for producing a C6 compound. In particular, the present invention relates to a recombinant microorganism, a recombinant protein, and a method for producing a C6 compound that can produce at least one of 2-hexenedioic acid and adipic acid. [Background technology]

[0002] Mono- and dicarboxylic acids such as adipic acid, succinic acid, fumaric acid, itaconic acid, and glutaric acid are important compounds in the chemical industry. For example, adipic acid (CAS No. 124-04-9) is a monomer compound that is used as a raw material for a variety of products, including synthetic fibers such as nylon-66, plastics, urethanes, and plasticizers.

[0003] Adipic acid is currently generally produced by nitric acid oxidation of cyclohexanol alone, which is derived from petroleum-derived benzene, or by nitric acid oxidation of a mixture of cyclohexanol and cyclohexanone. In the process, nitrous oxide (N 2 Alternatively, muconic acid can be reduced to adipic acid by chemical hydrogenation, which is a more environmentally friendly route than the traditional petrochemical route.

[0004] However, these chemical processes are energy intensive and are considered to be a cause of global warming. Therefore, a shift from fossil fuel-derived raw materials to renewable resource raw materials, including biomass-derived raw materials, is an important measure for realizing a low-carbon society.

[0005] Living organisms have various biosynthetic and metabolic pathways, and it is known that certain genetic engineering can be performed on microorganisms. Therefore, fermentation production using metabolically modified microorganisms by genetic engineering has attracted attention as a sustainable manufacturing method.

[0006] For example, microorganisms for the biosynthesis of adipic acid, 6-aminocaproic acid, or caprolactam have been reported (Patent Document 1). In this microbial method, adipic acid is produced from sugars using a transformant introduced with genes encoding "succinyl-CoA:acetyl-CoA acyltransferase" which converts succinyl-CoA and acetyl-CoA, which are biosynthesized in the citric acid cycle, to 3-oxoadipyl-CoA, "3-hydroxyacyl-CoA dehydrogenase" which converts 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, "3-hydroxyadipyl-CoA dehydratase" which converts 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA, "5-carboxy-2-pentenoyl-CoA reductase" which converts 5-carboxy-2-pentenoyl-CoA to adipyl-CoA, and "phosphotransadipylase" and "adipate kinase" which convert adipyl-CoA to adipate.

[0007] Microorganisms for the biosynthesis of muconic acid have also been reported (Non-Patent Document 1). In this microbial method, muconic acid is produced from sugars using a transformant introduced with genes encoding "3-dehydroshikimate dehydratase," which converts 3-dehydroshikimic acid synthesized via the shikimic acid pathway into protocatechuic acid, "protocatechuic acid decarboxylase," which converts protocatechuic acid into catechol, and "catechol 1,2-dioxygenase," which converts catechol into cis,cis-muconic acid.

[0008] Furthermore, a method has been reported in which a microorganism containing an enoate reductase is used to convert muconic acid into adipic acid via 2-hexenedioic acid (2,3-dehydroadipic acid), and then the muconic acid is reduced by chemical hydrogenation to obtain adipic acid, in order to replace the process with fermentation production (Patent Document 2).

[0009] Previously reported methods have room for improvement for industrial applications, and therefore there has been a demand for alternative methods to synthesize C6 compounds from biomass-derived carbon feedstocks. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Patent No. 5951990 specification [Patent Document 2] International Publication No. 2017 / 004709 [Non-patent literature]

[0011] [Non-Patent Document 1] JP 2019-195330 A Summary of the Invention [Problem to be solved by the invention]

[0012] The object of the present invention is to provide a recombinant microorganism, a recombinant protein and a production method capable of producing C6 compounds. A further object of the present invention is to provide a recombinant microorganism, a recombinant protein and a production method capable of producing at least one of 2-hexenedioic acid and adipic acid. [Means for solving the problem]

[0013] As a result of intensive research, the present inventors have found that C6 compounds can be efficiently produced by culturing a recombinant microorganism in which a specific exogenous enzyme is expressed in a host microorganism. Specifically, they have found that 2-hexenedioic acid and adipic acid can be produced from muconic acid by expressing an exogenous enzyme having a reducing activity for at least one of muconic acid and 2-hexenedioic acid in a recombinant microorganism, and have thus achieved the present invention.

[0014] That is, the present invention provides the following: [1] A recombinant microorganism comprising an exogenous gene encoding a protein having a reducing activity, the reducing activity is at least one of an activity of reducing muconic acid to 2-hexenedioic acid and an activity of reducing 2-hexenedioic acid to adipic acid; The exogenous gene is selected from the group consisting of: (i) a DNA encoding a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28, 29 or 30; (ii) DNA encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 28, 29 or 30; (iii) DNA consisting of a polynucleotide sequence having 80% or more sequence identity to the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60; (iv) DNA encoding a protein having an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence of the protein encoded by the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60; (v) a DNA that hybridizes under stringent conditions with a DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60; or (vi) DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60 a recombinant microorganism; [2] The exogenous gene is any one of the following: (vii) a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 28, 29 or 30, or (viii) DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60 [1] A recombinant microorganism according to [1], [3] The recombinant microorganism according to [1] or [2], wherein the exogenous gene is derived from at least one selected from Lachnospiraceae bacterium, Clostridium akagii, and Treponema saccharophilum; [4] The recombinant microorganism has a production pathway for C6 compounds, The recombinant microorganism according to any one of [1] to [3], wherein the C6 compound is at least one selected from the group consisting of adipic acid and 2-hexenedioic acid; [5] The recombinant microorganism according to any one of [1] to [4], wherein the recombinant microorganism belongs to the genus Escherichia, Bacillus, Corynebacterium, Arthrobacter, Brevibacterium, Clostridium, Zymomonas, Pseudomonas, Burkholderia, Streptomyces, Rhodococcus, Synechocystis, Alkalihalobacillus, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, or Aspergillus; [6] The recombinant microorganism according to any one of [1] to [5], wherein the recombinant microorganism is Escherichia coli; [7] The recombinant microorganism according to any one of [1] to [6], further comprising at least one selected from the group consisting of a gene encoding 2-enoyl-CoA reductase, a gene encoding 2,4-dienoyl-CoA reductase, a gene encoding FAD-dependent oxidoreductase, and a gene encoding NADH-flavin oxidoreductase / NADH oxidase; [8] A method for producing a C6 compound using the recombinant microorganism according to any one of [1] to [7], The production method, wherein the C6 compound is at least one selected from the group consisting of adipic acid and 2-hexenedioic acid; [9] (a) having at least one reducing activity of reducing muconic acid to 2-hexenedioic acid and reducing 2-hexenedioic acid to adipic acid; (b) Below: (i) an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28, 29 or 30; (ii) an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 28, 29 or 30; (iii) encoded by DNA consisting of a polynucleotide sequence having 80% or more sequence identity to the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60; (iv) an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence of the protein encoded by the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60; (v) encoded by DNA that hybridizes under stringent conditions to DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence set forth in SEQ ID NO: 58, 59 or 60; or (vi) a recombinant protein encoded by DNA consisting of a degenerate isomer of the polynucleotide sequence set forth in SEQ ID NO: 58, 59, or 60;

[10] (vii) consisting of the amino acid sequence shown in SEQ ID NO: 28, 29 or 30; or (viii) a recombinant protein according to [9], encoded by DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60;

[11] A method for producing a C6 compound using the recombinant protein according to [9] or

[10] , The method for producing said C6 compound is at least one selected from the group consisting of adipic acid and 2-hexenedioic acid. Effect of the Invention

[0015] According to the present invention, it is possible to provide a recombinant microorganism, a recombinant protein, and a production method capable of producing a C6 compound. Further, according to the present invention, it is possible to provide a recombinant microorganism, a recombinant protein, and a production method capable of producing at least one of 2-hexenedioic acid and adipic acid.

Brief Description of Drawings

[0016] [Figure 1] Figure 1 shows an example of the biosynthetic pathway from muconic acid to adipic acid. [Diagram 2] Figure 2 shows the test results of adipic acid production by a candidate protein of muconate reductase. [Diagram 3] Figure 3 shows the results of the fermentation production test of a muconic acid-producing bacterium. [Figure 4] Figure 4 shows the conversion activity from muconic acid to adipic acid based on the cell turbidity. [Diagram 5] Figure 5 shows the conversion activity from muconic acid to adipic acid based on the reaction time. [Figure 6] Figure 6 shows the protein used as a query and the selected candidate protein. [Figure 7] Figure 7 shows the amino acid sequences of the protein used as a query and the selected candidate protein. [Figure 8] Figure 8 shows the protein used as a query and the nucleotide sequence of the selected protein. [Figure 9] Figure 9 shows the nucleotide sequences of the primers for each protein.

Modes for Carrying Out the Invention

[0017] Embodiments of the present invention will be described in detail below. Note that the present invention is not limited to the following embodiments and can be implemented with various modifications within the scope of its gist. In addition, gene manipulations such as DNA acquisition, vector preparation, and transformation described in this specification can be performed by methods described in known documents such as Molecular Cloning 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience), and Experimental Notes on Genetic Engineering (Yodosha, Takaki Tamura), unless otherwise specified. Unless otherwise specified in this specification, nucleotide sequences are described from the 5'-end to the 3'-end. In this specification, the terms "polypeptide" and "protein" are used interchangeably.

[0018] In this specification, a numerical range indicated by "~" indicates a range including the numerical values described before and after "~" as the minimum value and the maximum value, respectively. In the numerical ranges described stepwise in this specification, the upper limit value or the lower limit value of a certain stepwise numerical range can be arbitrarily combined with the upper limit value or the lower limit value of another stepwise numerical range.

[0019] In this specification, the term "endogenous" or "endogenous nature" is used to mean that a host microorganism without modification by the gene recombination mentioned has the gene or protein (typically an enzyme) encoded thereby, regardless of whether it is functionally expressed to such an extent that it can promote a dominant biochemical reaction within the host cell.

[0020] As used herein, the terms "foreign" or "exogenous" are used to mean introducing a gene or nucleic acid sequence according to the present invention into a host when the host microorganism before genetic recombination does not have the gene to be introduced according to the present invention, does not substantially express the enzyme encoded by the gene, and although the gene or a different gene encodes the amino acid sequence of the enzyme, it does not express an endogenous enzyme activity comparable after genetic recombination.

[0021] The genetically modified microorganism according to the present invention contains an exogenous gene encoding a protein having a reducing activity. The "genetically modified microorganism" is also simply referred to as a "recombinant microorganism". Specifically, the above-mentioned reducing activity is: · Activity of reducing muconic acid to convert it into 2-hexenedioic acid (Activity A), and · Activity of reducing 2-hexenedioic acid to convert it into adipic acid (Activity B) is at least one of them. The reducing activity preferably includes both Activity A and Activity B.

[0022] Here, an example of the C6 compound production pathway of the recombinant microorganism according to the present invention, that is, the biosynthetic pathway from muconic acid to adipic acid, is shown in FIG. 1. Muconic acid is converted into 2-hexenedioic acid, and 2-hexenedioic acid is converted into adipic acid. That is, this production pathway consists of a two-step reaction of the reduction reaction of muconic acid and the reduction reaction of 2-hexenedioic acid, and includes a reaction of reducing muconic acid to convert it into 2-hexenedioic acid and a reaction of reducing 2-hexenedioic acid to convert it into adipic acid. Examples of the enzyme that can catalyze this conversion include polypeptides having the amino acid sequences shown in SEQ ID NOs: 1 to 30, and these polypeptides are each encoded by the nucleotide sequences shown in SEQ ID NOs: 31 to 60 (FIG. 8).

[0023] "A protein having at least one reducing activity of "activity A" which reduces muconic acid to convert it into 2-hexenedioic acid and "activity B" which reduces 2-hexenedioic acid to convert it into adipic acid" is also referred to as "muconate reductase". Further, the enzymatic activity of reducing muconic acid to convert it into adipic acid is also referred to as "muconate reductase activity" or "muconate reductase enzymatic activity", and these terms are used interchangeably.

[0024] In the present specification, "muconate reductase" is a polypeptide capable of performing a reduction reaction using a 2-enoic acid compound as a substrate under anaerobic conditions. Therefore, "muconate reductase activity" more specifically means an activity capable of promoting a reduction reaction using a 2-enoic acid compound as a substrate under anaerobic conditions.

[0025] In the present specification, muconic acid includes cis,cis-muconic acid, cis,trans-muconic acid, and trans,trans-muconic acid, and refers to one or more compounds selected from the group consisting of these compounds. Further, in the present specification, 2-hexenedioic acid includes cis-2-hexenedioic acid and trans-2-hexenedioic acid, and refers to one or more compounds selected from the group consisting of these compounds.

[0026] Here, regarding the sequence, the foreign gene contained in the recombinant microorganism according to the present invention is any of the following: (i) DNA encoding a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28, 29, or 30, (ii) DNA encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence shown in SEQ ID NO: 28, 29, or 30, (iii) DNA consisting of a polynucleotide sequence having 80% or more sequence identity with the polynucleotide sequence shown in SEQ ID NO: 58, 59, or 60, (iv) DNA encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added with respect to the amino acid sequence of the protein encoded by the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, (v) DNA that hybridizes under stringent conditions with DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, and (vi) DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60.

[0027] Regarding the sequence, the foreign gene contained in the recombinant microorganism according to the present invention is preferably as follows: (i) DNA encoding a protein consisting of an amino acid sequence having 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28, 29 or 30, (ii) DNA encoding a protein consisting of an amino acid sequence in which 1 to 7, 1 to 5 or 1 to 3 amino acids are deleted, substituted, inserted and / or added with respect to the amino acid sequence shown in SEQ ID NO: 28, 29 or 30, (iii) DNA consisting of a polynucleotide sequence having 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more or 99% or more sequence identity with the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, (iv) DNA encoding a protein consisting of an amino acid sequence in which 1 to 7, 1 to 5 or 1 to 3 amino acids are deleted, substituted, inserted and / or added with respect to the amino acid sequence of the protein encoded by the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, (v) DNA that hybridizes under stringent conditions with DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, or (vi) DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60 is.

[0028] Regarding the array, the foreign gene contained in the recombinant microorganism according to the present invention is more preferably as follows: (vii) DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 28, 29 or 30, or (viii) DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60 That's it.

[0029] The genetically modified microorganism of the present invention contains a foreign gene encoding a protein having at least one reducing activity (i.e., muconic acid reductase activity) of the activity (activity A) of reducing muconic acid to convert it into 2-hexenedioic acid and the activity (activity B) of reducing 2-hexenedioic acid to convert it into adipic acid. Therefore, the enzyme (i.e., muconic acid reductase) for the reaction in FIG. 1 is expressed.

[0030] Here, the reductase gene contained in the recombinant microorganism according to the prior art is compared with the above foreign gene contained in the recombinant microorganism according to the present invention from the viewpoint of sequence identity. That is, the reductase gene contained in the recombinant microorganism according to the prior art and the foreign gene of the reductase contained in the recombinant microorganism according to the present invention have a sequence identity of 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more or 80% or more in the nucleotide sequence. Also, the reductase contained in the recombinant microorganism according to the prior art and the above reductase contained in the recombinant microorganism according to the present invention have a sequence identity of 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more or 80% or more in the amino acid sequence.

[0031] For example, the nucleotide sequence identity between the prior art reductase gene having the nucleotide sequence shown in SEQ ID NO: 31 and the exogenous gene of the reductase contained in the recombinant microorganism according to the present invention is 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more in the nucleotide sequence. Also, the nucleotide sequence identity between the prior art reductase gene having the nucleotide acid sequence shown in SEQ ID NO: 32 and the exogenous gene of the reductase contained in the recombinant microorganism according to the present invention is 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more in the nucleotide sequence. Here, SEQ ID NOs: 31 and 32 are the nucleotide sequences encoding NADH flavin oxidoreductase / NADH oxidase derived from Weizmannia coagulans 36D1 and the nucleotide sequence encoding 2-enoyl reductase derived from Clostridium acetobutylicum DSM 1731, respectively.

[0032] For example, the prior art reductase having the amino acid sequence shown in SEQ ID NO: 1 and the reductase contained in the recombinant microorganism according to the present invention have 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, or 80% or more sequence identity in the amino acid sequence. Further, the prior art reductase having the amino acid sequence shown in SEQ ID NO: 2 and the reductase contained in the recombinant microorganism according to the present invention have 65% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, or 78% or more sequence identity in the amino acid sequence. Here, SEQ ID NOs: 1 and 2 are the amino acid sequences of NADH flavin oxidoreductase / NADH oxidase derived from Weizmannia coagulans 36D1 and the amino acid sequence of 2-enoyl reductase derived from Clostridium acetobutylicum DSM 1731, respectively.

[0033] The foreign gene contained in the recombinant microorganism according to the present invention is derived from at least one selected from Lachnospiraceae bacterium, Clostridium akagii, and Treponema saccharophilum.

[0034] By containing the above foreign gene, the recombinant microorganism has good muconic acid reductase activity, and by using the recombinant microorganism having such enzyme activity, the target compound, a C6 compound, can be produced. By containing the above foreign gene, it is possible to produce the target compound in a good yield.

[0035] It is understood by those skilled in the art that the "C6 compound" in the present specification can take a neutral or ionized form including any salt form, and that this form is pH-dependent.

[0036] In the present specification, the "C6 compound" includes · Adipic acid (CAS No. 124-04-9), and · 2-Hexenedioic acid (CAS No. 4440-68-0) are included, and the "C6 compound" refers to one or more compounds selected from the group consisting of these compounds. That is, the C6 compound is at least one selected from adipic acid and 2-hexenedioic acid.

[0037] The microorganism of the present invention is a genetically modified microorganism into which an exogenous enzyme gene has been introduced as described above. The "genetically modified microorganism" is also simply referred to as the "recombinant microorganism".

[0038] In one aspect, the recombinant microorganism according to the present invention has a production pathway for a C6 compound, and the C6 compound is at least one selected from the group consisting of adipic acid and 2-hexenedioic acid.

[0039] In this specification, with respect to a certain compound, "having a production pathway" means that the genetically modified microorganism according to the present invention expresses an amount of enzyme sufficient for each reaction step of the production pathway of the compound to proceed, and can biosynthesize the compound. The recombinant microorganism of the present invention may be one using a host microorganism that originally has the ability to produce the compound, or may be one obtained by modifying a host microorganism that originally does not have the ability to produce the compound so as to have the ability to produce the compound.

[0040] In this specification, "derived from" means that the gene or the protein (typically an enzyme) encoded thereby is intrinsically possessed by the specific species mentioned.

[0041] In connection with the present invention, the host microorganism into which the target foreign gene is introduced is not particularly limited and may be either a prokaryote or a eukaryote. It can be arbitrarily selected from those already isolated and preserved, those newly isolated from nature, and those genetically modified. The host microorganism belongs to, for example, the genus Escherichia, Bacillus, Corynebacterium, Arthrobacter, Brevibacterium, Clostridium, Zymomonas, Pseudomonas, Burkholderia, Streptomyces, Rhodococcus, Synechocystis, Alkalihalobacillus, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, or Aspergillus. The host microorganism preferably belongs to the genus Escherichia, and more preferably is Escherichia coli.

[0042] Regarding the conditions for promoting the muconic acid reduction reaction according to the present invention, any conditions under which the target product is produced may be used, and those skilled in the art can adjust and set the composition, pH, reaction temperature, reaction time, etc. of the reaction solution by the methods commonly used. For example, as the pH of the reaction solution, a buffer solution with a pH of 7 to 9 can be mentioned, and as more suitable conditions, a buffer solution with a pH of 8 to 9 can be mentioned. The reaction temperature is usually 20 to 40 °C, and more preferably 30 to 37 °C. The reaction time may be any time during which the target product can be produced. When the culture of the recombinant microorganism according to the present invention is mixed with the substrate compound, for example, it is 1 hour to 7 days. When the recombinant polypeptide according to the present invention is extracted from the culture of the recombinant microorganism and mixed with the substrate compound, for example, it is 15 minutes to 48 hours.

[0043] Since the muconate reductase according to the present invention has an Fe-S cluster, it preferably reacts under anaerobic conditions. Further, in order to activate the Fe-S cluster inactivated by structural changes, it is preferable to add an electron donor. When a culture of the recombinant microorganism according to the present invention is mixed with a substrate compound, for example, glucose, when the recombinant polypeptide according to the present invention is extracted from the culture of the recombinant microorganism and mixed with the substrate compound, for example, nicotinamide adenine dinucleotide phosphate (NADPH) is preferably added.

[0044] In one aspect, the recombinant microorganism according to the present invention further comprises at least one selected from the group consisting of a gene encoding 2-enoyl-CoA reductase, a gene encoding 2,4-dienoyl-CoA reductase, a gene encoding an FAD-dependent oxidoreductase, and a gene encoding NADH flavin oxidoreductase / NADH oxidase. By further expressing an enzyme that catalyzes the reaction steps of the C6 compound production pathway, the recombinant microorganism according to the present invention can produce various C6 compounds.

[0045] The recombinant microorganism according to the present invention may be one in which any gene is appropriately disrupted in the host microorganism. Disruption of the target gene is performed by a method known in the art.

[0046] In addition, when the genetically modified microorganism of the present invention expresses a sufficient amount of an enzyme that catalyzes the reaction steps of the C6 compound production pathway without introduction of a foreign gene, the reaction may proceed with the enzyme encoded by the endogenous gene.

[0047] Therefore, the second aspect of the present invention relates to a polypeptide, specifically a recombinant protein, having a reducing activity which is at least one of an activity of reducing muconic acid to convert it into 2-hexenedioic acid and an activity of reducing 2-hexenedioic acid to convert it into adipic acid.

[0048] The recombinant protein according to the present invention is an enzyme derived from, for example, at least one of the following species: Lachnotalea glycerini (SEQ ID NO: 11), Propionispira arboris (SEQ ID NO: 12), Sarcina ventriculi (SEQ ID NO: 13), Clostridium sp. (SEQ ID NO: 14), Clostridium sp. 001 (SEQ ID NO: 15), Fusibacter paucivorans (SEQ ID NO: 16), Anaerovorax odorimutans (SEQ ID NO: 17), Halocella sp. SP3-1 (SEQ ID NO: 18), Clostridium algoriphilum (SEQ ID NO: 19), Caloramator sp. E03 (SEQ ID NO: 20), Sporomusa acidovorans (SEQ ID NO: 21), Eubacterium sp. (SEQ ID NO: 22), Pelosinus propionicus (SEQ ID NO: 23), Firmicutes bacterium (SEQ ID NO: 24), Propionispora vibrioides (SEQ ID NO: 25), Ruminiclostridium papyrosolvens (SEQ ID NO: 26), Orenia metallireducens (SEQ ID NO: 27), Lachnospiraceae bacterium (SEQ ID NO: 28), Clostridium akagii (SEQ ID NO: 29), and Treponema saccharophilum (SEQ ID NO: 30).

[0049] The recombinant protein according to the present invention is preferably derived from at least one of Pelosinus propionicus (SEQ ID NO: 23), Firmicutes bacterium (SEQ ID NO: 24), Propionispora vibrioides (SEQ ID NO: 25), Ruminiclostridium papyrosolvens (SEQ ID NO: 26), Orenia metallireducens (SEQ ID NO: 27), Lachnospiraceae bacterium (SEQ ID NO: 28), Clostridium akagii (SEQ ID NO: 29) and Treponema saccharophilum (SEQ ID NO: 30), more preferably derived from at least one of Lachnospiraceae bacterium (SEQ ID NO: 28), Clostridium akagii (SEQ ID NO: 29) and Treponema saccharophilum (SEQ ID NO: 30).

[0050] The recombinant protein according to the present invention is: (a) It has a reducing activity which is at least one of the activities of reducing muconic acid to convert it into 2-hexenedioic acid and reducing 2-hexenedioic acid to convert it into adipic acid, (b) The following: (i) It consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28, 29 or 30, (ii) It consists of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 28, 29 or 30, (iii) It is encoded by DNA consisting of a polynucleotide sequence having 80% or more sequence identity with the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, (iv) It consists of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of the protein encoded by the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, (v) DNA that hybridizes under stringent conditions with a DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence shown in SEQ ID NO: 58, 59, or 60, or (vi) encoded by a DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 58, 59, or 60.

[0051] The recombinant protein according to the present invention is preferably: (a) having at least one reducing activity, which is the activity of reducing muconic acid to convert it into 2-hexenedioic acid and the activity of reducing 2-hexenedioic acid to convert it into adipic acid, (b) the following: (i) consisting of an amino acid sequence having 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28, 29, or 30, (ii) consisting of an amino acid sequence in which 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence shown in SEQ ID NO: 28, 29, or 30, (iii) encoded by a DNA consisting of a polynucleotide sequence having 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the polynucleotide sequence shown in SEQ ID NO: 58, 59, or 60, (iv) consisting of an amino acid sequence in which 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence of the protein encoded by the polynucleotide sequence shown in SEQ ID NO: 58, 59, or 60, (v) encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence shown in SEQ ID NO: 58, 59, or 60, or (vi) encoded by a DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 58, 59, or 60.

[0052] The recombinant protein according to the present invention is more preferably: (a) having a reducing activity which is at least one of an activity of reducing muconic acid to convert it into 2-hexenedioic acid and an activity of reducing 2-hexenedioic acid to convert it into adipic acid, (b) the following: (vii) consisting of the amino acid sequence shown in SEQ ID NO: 28, 29 or 30, or (viii) encoded by DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60.

[0053] The recombinant protein according to the present invention more preferably has both reducing activities of an activity of reducing muconic acid to convert it into 2-hexenedioic acid and an activity of reducing 2-hexenedioic acid to convert it into adipic acid.

[0054] The gene encoding the recombinant protein according to the present invention may be derived from other than the exemplified organisms or may be artificially synthesized, as long as it can express substantial enzyme activity in host microbial cells.

[0055] Also, the amino acid sequence of the recombinant protein according to the present invention, or the base sequence of the gene encoding the enzyme, may have all mutations that can occur in nature, as well as artificially introduced mutations and modifications, as long as they can express substantial enzyme activity in the host microbial cells. For example, mutations such as deletions, substitutions, insertions, and additions may be contained. For example, it may contain an amino acid sequence in which one or more, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 7, even more preferably 1 to 5, and particularly preferably 1 to 3 amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence of the above enzyme.

[0056] Furthermore, as long as the nucleotide sequence of the gene encoding the recombinant protein according to the present invention can express substantial enzyme activity in the host microbial cell, DNA having a complementary nucleotide sequence and DNA that hybridizes with it under stringent conditions can also be used. The "stringent conditions" are, for example, conditions such as "1xSSC, 0.1% SDS, 55°C" which are the washing conditions for Southern hybridization, and more stringent conditions are conditions such as "0.1xSSC, 0.1% SDS, 60°C".

[0057] In addition, it is known that there are redundant codons for various codons encoding specific amino acids. Therefore, alternative codons that will ultimately be translated into the same amino acid may also be used in the present invention. That is, since the genetic code is degenerate, multiple codons can be used to encode a specific amino acid, and thus the amino acid sequence can be encoded by any set of similar DNA oligonucleotides. Since most organisms are known to preferentially use a subset of specific codons (optimal codons) (Gene, Vol. 105, pp. 61-72, 1991, etc.), performing "codon optimization" according to the host microorganism may also be useful in the present invention.

[0058] Therefore, the genetically modified microorganism according to the present invention may include a nucleotide sequence having a sequence identity of, for example, 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more with the nucleotide sequence of the above enzyme gene, on the condition that it can express enzyme activity. Alternatively, it may include a gene encoding a protein containing an amino acid sequence having a sequence identity of, for example, 80% or more, 85% or more, 88% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence of the above enzyme.

[0059] In the present specification, the percentage (%) of "sequence identity" of a comparative amino acid sequence with respect to a reference amino acid sequence is defined as the percentage of amino acid residues in the comparative sequence that are identical to the amino acid residues in the reference sequence when the sequences are aligned so that the identity between the two sequences is maximized and gaps are introduced into one or both of the two sequences if necessary. At this time, conservative substitutions are not considered as part of the sequence identity. The sequence identity can be determined by using publicly available computer software, for example, it can be determined by using an alignment search tool such as BLAST (registered trademark, hereinafter omitted) (Basic Local Alignment Search Tool). A person skilled in the art can determine appropriate parameters for obtaining the maximum alignment of the comparative sequence in the alignment. The "sequence identity" of a nucleotide sequence can also be determined by a similar method.

[0060] In the present invention, by introducing the above C6 compound biosynthetic enzyme gene into a host microbial cell as an "expression cassette", a more stable and high-level enzyme activity can be obtained. In the present specification, the "expression cassette" means a nucleotide containing a nucleic acid sequence that regulates transcription and translation functionally linked to a nucleic acid or gene to be expressed. Typically, the expression cassette of the present invention contains a promoter sequence functionally linked 5' upstream from the coding sequence, a terminator sequence 3' downstream, and optionally further normal regulatory elements, and in such a case, the nucleic acid or gene to be expressed is introduced into a host microorganism.

[0061] A promoter is defined as a DNA sequence that binds RNA polymerase to DNA and initiates RNA synthesis, regardless of whether it is a constitutive expression promoter or an inducible expression promoter. A strong promoter is a promoter that initiates mRNA synthesis at a high frequency and is also preferably used in the present invention. In Escherichia coli, the lac system, trp system, tac or trc system, the major operator and promoter regions of λ phage, the control region of fd coat protein, promoters for glycolytic enzymes (e.g., 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase), glutamate decarboxylase A, serine hydroxymethyltransferase, the promoter region of T7 phage-derived RNA polymerase, etc. can be used. In Corynebacterium glutamicum, the HCE (high-level constitutive expression) promoter, cspB promoter, sodA promoter, elongation factor (EF-Tu) promoter, etc. can be used. As terminators, the T7 terminator, rrnBT1T2 terminator, lac terminator, etc. can be used. In addition to promoter and terminator sequences, examples of other regulatory elements that can be mentioned are selection markers, amplification signals, origins of replication, etc. Suitable regulatory sequences are described, for example, in "Gene Expression Technology: Methods in Enzymology 185," Academic Press (1990).

[0062] The expression cassette described above is incorporated into a vector consisting of, for example, a plasmid, phage, transposon, IS element, fosmid, cosmid, or linear or circular DNA, etc., and inserted into a host microorganism. Plasmids and phages are preferred. These vectors may be autonomously replicated in the host microorganism or replicated by the chromosome. Suitable plasmids include, for example, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11 or pBdCI of Escherichia coli; pUB110, pC194 or pBD214 of Bacillus; pSA77 or pAJ667 of Corynebacterium. Examples of Bacillus such as Bacillus genus include pUB110, pC194 or pBD214. Other plasmids and the like that can be used are described in "Gene Cloning and DNA analysis 7th edition", Wiley-Blackwell (2016). Introduction of the expression cassette into the vector can be achieved by conventional methods including excision with appropriate restriction enzymes, cloning, and ligation. Each expression cassette may be arranged on one vector or on two or more vectors.

[0063] After constructing the vector having the expression cassette of the present invention as described above, conventional methods can be used when introducing the vector into a host microorganism. For example, the calcium chloride method, electroporation method, conjugation transfer method, protoplast fusion method, etc. can be mentioned, but it is not limited to these, and a method suitable for the host microorganism can be selected.

[0064] The third aspect of the present invention relates to a method for producing a C6 compound using the aforementioned recombinant microorganism or the aforementioned recombinant protein. When using a recombinant microorganism, the production method includes a culturing step of culturing the recombinant microorganism.

[0065] Here, the C6 compound is preferably at least one selected from the group consisting of adipic acid and 2-hexenedioic acid.

[0066] The method for producing a C6 compound includes a culturing step of culturing the genetically modified microorganism according to the present invention. In the culturing step, the recombinant microorganism is cultured in a medium containing a carbon source and a nitrogen source to obtain a culture containing cells. The genetically modified microorganism of the present invention is cultured under conditions suitable for C6 compound production and the growth and / or maintenance of the microorganism, and suitable medium composition, culturing time, and culturing conditions can be easily set by those skilled in the art.

[0067] Examples of the carbon source include D-glucose, sucrose, lactose, fructose, maltose, oligosaccharide, polysaccharide, starch, cellulose, rice bran, molasses, oils and fats (such as soybean oil, sunflower oil, peanut oil, coconut oil, etc.), fatty acids (such as palmitic acid, linoleic acid, oleic acid, linolenic acid, etc.), alcohols (such as glycerol, ethanol, etc.), organic acids (such as acetic acid, lactic acid, succinic acid, etc.), corn hydrolysis solution, and cellulose hydrolysis solution. Preferably, it is D-glucose, sucrose or glycerol. These carbon sources can be used individually or as a mixture.

[0068] The C6 compound produced using a biomass-derived raw material can be clearly distinguished from synthetic raw materials derived from, for example, petroleum, natural gas, coal, etc. by measuring the bio-based carbon content based on the Carbon-14 (radioactive carbon) analysis defined in ISO16620-2 or ASTM D6866.

[0069] Examples of nitrogen sources include nitrogen-containing organic compounds (such as peptone, casamino acid, tryptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean powder, amino acids, and urea), or inorganic compounds (such as aqueous ammonia solution, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, sodium nitrate, ammonium nitrate, etc.). These nitrogen sources can be used individually or as a mixture.

[0070] In addition, when the recombinant microorganism expresses a useful additional trait, for example, when it has a resistance marker to an antibiotic, the medium may contain the corresponding antibiotic. Thereby, the risk of contamination by miscellaneous bacteria during culture is reduced. Examples of antibiotics include, but are not limited to, β-lactam antibiotics such as ampicillin, aminoglycoside antibiotics such as kanamycin, macrolide antibiotics such as erythromycin, tetracycline antibiotics, chloramphenicol, etc.

[0071] The culture may be in batch mode or continuous mode. Also, in any case, it may be in a form of replenishing the additional carbon source etc. at an appropriate time during the culture. Furthermore, the culture may be carried out while controlling conditions such as suitable temperature, oxygen concentration, pH, etc. The suitable culture temperature for transformants derived from common microbial host cells is usually in the range of 15°C to 55°C, preferably 25°C to 40°C. When the host microorganism is aerobic, shaking (such as flask culture etc.) or stirring / aeration (such as jar fermenter culture etc.) may be carried out to ensure an appropriate oxygen concentration during fermentation. Those culture conditions can be easily set by those skilled in the art.

[0072] The culturing step may include obtaining a culture of the recombinant microorganism and / or an extract of the culture.

[0073] The method for producing the compound according to the present invention may further include a mixing step of mixing the culture and / or the extract of the culture with a substrate compound to obtain a mixed solution. The substrate compound may be appropriately selected according to the enzyme and the target compound.

[0074] The production method according to the present invention may include a step of separating and / or purifying the target compound, i.e., the C6 compound. In the step of separating and / or purifying, any method can be used, for example, centrifugation, membrane filtration, membrane separation, crystallization, extraction, distillation, adsorption, phase separation, ion exchange, and various chromatography methods, but it is not limited thereto. In the separation and / or purification step, one method may be selected and implemented, or a plurality of methods may be combined and implemented.

[0075] As described above, according to the present invention, by introducing an exogenous gene encoding a protein having the muconic acid reductase activity into a host microorganism, a recombinant microorganism capable of producing at least one of adipic acid and 2-hexenedioic acid from muconic acid can be obtained. In addition, a recombinant protein capable of producing at least one of adipic acid and 2-hexenedioic acid from muconic acid can also be provided.

[0076] In addition, depending on the desired C6 compound, various C6 compounds can be obtained by performing conversion using additional enzymes.

[0077] In addition, the present invention can produce the target C6 compound in a good yield as compared with the biosynthesis of existing C6 compounds. In particular, for 2-hexenedioic acid and adipic acid, it is possible to produce them in a good yield as compared with the biosynthesis methods of the prior art.

[0078] In the prior art, although there are examples reported where C6 compounds can be biosynthesized with good yields, there are also cases where, due to a high concentration of cells in the reaction system and / or a low concentration of the substrate compound, a high yield is computationally obtained even though the conversion activity of the enzyme is not sufficiently high. For example, there are reports of biosynthesizing C6 compounds with good yields using a substrate compound at a concentration that is one-tenth of what is desired for industrial-scale production. However, because the concentration of the substrate compound is low, the concentration of the target compound produced is also low, and it is considered not suitable for industrial-scale use. In contrast, according to the present invention, even when the concentration of recombinant microorganisms and recombinant proteins in the reaction system is low, the reduction reaction can proceed to obtain 2-hexenedioic acid and adipic acid in good yields and at high concentrations of the target compound. Furthermore, the reduction reaction can proceed sufficiently even in a short reaction time to produce high concentrations of 2-hexenedioic acid and adipic acid. Thus, the recombinant microorganisms and recombinant proteins according to the present invention have a sufficiently high enzyme conversion activity, so that even when the concentration of the substrate compound is high relative to their concentration in the reaction system, the reduction reaction can proceed sufficiently to produce a high concentration of the target compound. That is, according to the present invention, it is possible to efficiently produce the target compound in a short time and with a high yield using a small amount of recombinant microorganisms or recombinant proteins, and thus it is considered to be fully suitable for use in the production of the target compound on an industrial scale.

[0079] Those skilled in the art given the above description can fully implement the present invention. Hereinafter, examples are given for the purpose of further explanation. Therefore, the present invention is not limited to these examples.

Examples

[0080] Hereinafter, the present invention will be described based on examples, but the present invention is not limited to these examples.

[0081] <1> Construction of an adipic acid-producing strain Using the 2-enoyl-CoA reductase sequences from Weizmannia coagulans 36D1 and Clostridium acetobutylicum DSM 1731 as queries, a homology search was performed by PROTEIN BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins), and the hit amino acid sequences were mapped onto a phylogenetic tree. As candidate enzymes presumed to have 2-enoyl-CoA reductase function, 21 proteins were selected from a clade different from the above amino acid sequences. Also, proteins annotated as enoate reductase were searched from the NCBI (http: / / www.ncbi.nlm.nih.gov / ) database, and the 1307 hit amino acid sequences were clustered by an original analysis method. As candidate enzymes presumed to have 2-enoyl-CoA reductase function, 8 proteins were selected from the same cluster as the 2-enoyl-CoA reductases from Weizmannia coagulans 36D1 and Clostridium acetobutylicum DSM 1731 among the 10 classified clusters. The proteins used as queries (enzyme numbers C1 and C2), as well as the selected candidate proteins (enzyme numbers ER1~ER28) are shown in Figure 6.

[0082] The polynucleotides encoding the 2-enoyl-CoA reductase from Weizmannia coagulans 36D1, the 2-enoyl-CoA reductase from Clostridium acetobutylicum DSM 1731, and the selected enoate reductase candidate proteins were optimized for E. coli expression using GENEius (software name, Eurofins), and obtained using the artificial gene synthesis service of Eurofins Genomics. The amino acid sequences and nucleotide sequences of the selected proteins are shown in Figures 7 and 8.

[0083] Using PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio Inc.), PCR was performed with the oligonucleotides shown in Table 4 as primers to obtain PCR products for each gene region. Next, using pACYCDuet-1 (product name, manufactured by Novagen) as a template, PCR was performed with the oligonucleotides shown in Figure 9 as primers to obtain a pRSFDuet-1 fragment. The DNA fragment containing each enoate reductase region and the pACYCDuet-1 fragment were ligated using the In-Fusion (registered trademark. hereinafter omitted) HD cloning kit (product name, manufactured by Clontech). It was transformed into Escherichia coli JM109 strain and cultured overnight at 37 °C on an LB agar medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, agar powder 15 g / L) containing 20 mg / L of chloramphenicol to obtain transformants. A plasmid was extracted from the obtained transformants to obtain an expression plasmid for the enoate reductase candidate protein.

[0084] Referring to Document A (Akhtar, M.K., Jones, P.R. Deletion of iscR stimulates recombinant clostridial Fe?Fe hydrogenase activity and H2-accumulation in Escherichia coli BL21(DE3). Appl Microbiol Biotechnol 78, 853?862 (2008).), an Escherichia coli BL21(DE3)ΔiscR strain in which the iscR gene of Escherichia coli BL21(DE3) strain competent cells (manufactured by Nippon Gene) was disrupted was obtained.

[0085] The plasmid constructed in Escherichia coli BL21(DE3)ΔiscR strain was transformed by the electroporation method and cultured overnight at 37 °C on an LB agar medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, agar powder 15 g / L) containing 20 mg / L of chloramphenicol to obtain transformants.

[0086] <2> Screening of muconate reductase The transformants C1, C2 and ER1 - ER28 were inoculated into 2 mL of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, agar powder 15 g / L) containing 20 mg / L of chloramphenicol (in a 14 mL round - bottom tube), and cultured with shaking overnight at 37°C and 200 rpm to obtain a preculture solution.

[0087] The obtained preculture solution was inoculated into 12 mL of MagicMedia (trademark) E. coli Expression Medium (product name, manufactured by Invitrogen) containing 20 mg / L of chloramphenicol (in a 15 mL conical tube), and cultured with shaking at 37°C and 120 rpm for 2 days.

[0088] After the culture was completed, the supernatant was discarded by centrifugation (5,000×g, 10 minutes) to obtain bacterial cells. The obtained bacterial cells were suspended using 200 mM phosphate buffer (pH 7.0) to an OD600 of 100.

[0089] 125 μL of the bacterial cell suspension, 5 μL of 3M glucose solution, 2.5 μL of 1M sodium chloride solution, and 5 μL of 10 g / L cis,cis - muconic acid solution were added to a microtube. The cis,cis - muconic acid used as the substrate was neutralized with sodium hydroxide to obtain a solution. 112.5 μL of ultrapure water was added to prepare a reaction solution with a total volume of 250 μL.

[0090] The prepared reaction solution was subjected to a shaking reaction at 37°C and 180 rpm in a thermostatic chamber. The reaction solution 24 hours after the start of the reaction was filtered through a hydrophilic PVDF filter (manufactured by Corning), and measured using ultra - high performance liquid chromatography - mass spectrometry (UPLC - MS; manufactured by Waters). Detector: TUV Detector (UV), SQ Detector2 (MS) Column: ACQUITY UPLC BEH C18 Column (130 Å, 1.7 μm, 2.1 mm×50 mm; manufactured by Waters) Mobile phase: Acetonitrile / Milli Q = 90 / 10

[0091] The results are shown in Figure 2 and Table 1. In the culture of transformant C1, 133 mg / L of adipic acid was produced, and in the culture of transformant C2, 133 mg / L of adipic acid was produced.

[0092] In the cultures of transformants ER1 to ER8, neither adipic acid nor the intermediate 2,3-dehydroadipic acid was produced.

[0093] In the cultures of transformants ER9 to ER28, adipic acid and the intermediate 2,3-dehydroadipic acid were produced. From this result, it was shown that the protein shown above has muconate reductase activity.

[0094] Among the above, in the cultures of transformants ER26, ER27, and ER28 (corresponding to Examples 1, 2, and 3, respectively), 148 mg / L, 179 mg / L, and 181 mg / L of adipic acid were produced, respectively, showing productivity higher than that of other transformants. Among them, in transformants C1 and C2, 40 mg to 50 mg of the intermediate accumulated, while in transformant ER28, only 5 mg of the intermediate accumulated, and the conversion rate from muconic acid to adipic acid was 97.4%. From this result, Examples 1 to 3 were shown to have good enzyme activity for promoting the reduction reaction of muconic acid.

[0095]

Table 1

[0096] <3> Fermentative production of muconic acid Referring to Non-Patent Document 1 (Japanese Patent Laid-Open No. 2019-195330), the metabolic modification of Escherichia coli BL21(DE3) strain competent cells (manufactured by Nippon Gene Co., Ltd.) was carried out to obtain a cis,cis-muconic acid-producing strain.

[0097] The above strain was inoculated into 2 mL of LB liquid medium (this medium contains 10 g / L of tryptone, 5 g / L of yeast extract, 5 g / L of sodium chloride, and 15 g / L of agar powder) containing 50 mg / L of kanamycin sulfate (14 mL round-bottom tube), and cultured with shaking overnight at 37 °C and 200 rpm to obtain a preculture solution.

[0098] The obtained preculture solution was inoculated into 100 mL of MM medium (Table 2) containing 50 mg / L of kanamycin sulfate, 0.1 M of IPTG, 40 g / L of glucose, and 10% of LB liquid medium (10 g / L of tryptone, 5 g / L of yeast extract, 5 g / L of sodium chloride, 15 g / L of agar powder) (100 mL jar culture device; Bio Jr.8, manufactured by Biott) for main culture. The culture conditions were: culture temperature: 37 °C, culture pH: 7.0, alkali addition: 10% aqueous ammonia, stirring speed: 700 rpm, aeration rate: 0.1 mL / min.

[0099]

Table 2

[0100] The above culture solution was centrifuged at 10,000 g for 3 minutes to recover the supernatant, filtered through a hydrophilic PVDF filter (manufactured by Corning), and measured using high-performance liquid chromatography (HPLC; manufactured by HITACHI). Detector: L-2400 (UV), L-2490 (RI) Column: Shim-Pack Fast-OA(G) (4.0 mm × 10 mm; manufactured by Shimadzu), Fast-OA, Fast-OA (connected; 7.8 mm × 10 mm; manufactured by Shimadzu) Mobile phase: 8 mM methanesulfonic acid aqueous solution

[0101] The concentration of cis,cis-muconic acid (g / L), glucose concentration (g / L), and cell density (OD600) after culture are shown in Figure 3. The medium after 120 hours of culture was colored brown and produced 10.1 g / L of muconic acid.

[0102] <4>Evaluation of adipic acid production ability from the fermentation broth of muconic acid The transformant C1 and ER28 strain were inoculated into 2 mL of LB liquid medium containing 20 mg / L of chloramphenicol (the medium contains 10 g / L of tryptone, 5 g / L of yeast extract, 5 g / L of sodium chloride, and 15 g / L of agar powder) (14 mL round-bottom tube), and cultured with shaking overnight at 37 °C and 200 rpm to obtain a preculture solution.

[0103] The obtained preculture solution was inoculated into 250 mL of MagicMedia (trademark) E. coli Expression Medium (product name, manufactured by Invitrogen) containing 20 mg / L of chloramphenicol (300 mL bottle), and cultured with shaking overnight at room temperature and 130 rpm (microaerobic condition).

[0104] After the completion of the culture, the supernatant was discarded by centrifugation (5,000×g, 10 minutes) to obtain the cells. The obtained cells were suspended using 1 mL of the muconic acid fermentation broth obtained in <3> above so that the OD600 became 20 and 100.

[0105] Glucose was added to the prepared reaction solution to a concentration of 40 g / L, and the reaction was carried out with shaking at 37 °C and 180 rpm in a constant temperature chamber. The reaction solution was sampled 30 hours after the start of the reaction and measured using high performance liquid chromatography (HPLC; manufactured by HITACHI) in the same manner as in <3> above.

[0106] The results are shown in Figures 4 and 3. The concentration of muconic acid at the start of the reaction was 6.5 g / L due to dilution by the cells.

[0107] The transformant C1 did not produce adipic acid at OD20, but produced 2.7 g / L of adipic acid at OD100. The conversion rates after 30 hours of reaction were 0.0% and 41.5% respectively.

[0108] The transformant ER28 produced 1.7 g / L of adipic acid at OD20 and 5.3 g / L at OD100. The conversion rate after 30 hours of reaction was 82.1%. It had muconate reductase activity even at low cell turbidity and produced adipic acid well at high cell turbidity.

[0109]

Table 3

[0110] <5>Evaluation of the ability to produce adipic acid from the muconic acid fermentation broth The cells obtained in <4> above were suspended in a microtube using 2 mL of the muconic acid fermentation broth obtained in <3> above. The cell density after suspension was as shown in Table 4.

[0111]

Table 4

[0112] Glucose was added to the prepared reaction solution to a concentration of 20 g / L, and the reaction was carried out with shaking at 37°C and 180 rpm in a constant temperature chamber. In addition, 100 μL of a 500 g / L glucose solution was added 4 hours after the start of the reaction. The reaction solutions were sampled 3 hours, 6 hours, and 23 hours after the start of the reaction and measured using high performance liquid chromatography (HPLC; manufactured by HITACHI) in the same manner as in <3> above.

[0113] The results are shown in Figure 5 and Table 5. Note that due to dilution by the cells, the concentration of muconic acid at the start of the reaction was 6.5 g / L. In transformant C1, 1.5 g / L of adipic acid was produced in 3 hours of reaction, and the production amount of adipic acid was not affected by the passage of time. The conversion rate after 3 hours of reaction was 22.6%.

[0114] In transformant ER28, 5.4 g / L of adipic acid was produced in 3 hours of reaction. The conversion rate after 23 hours of reaction was 83.9%, and adipic acid was produced well even in a short reaction time.

[0115] TIFF2025087535000006.tif27163 [Industrial Applicability]

[0116] According to the present invention, a biosynthetic process of a C6 compound can be provided, and this process is expected to be applied to industrial-scale production.

Claims

1. A recombinant microorganism comprising a foreign gene encoding a protein having reducing activity, wherein the reducing activity is at least one of an activity of reducing muconic acid to convert it into 2-hexenedioic acid and an activity of reducing 2-hexenedioic acid to convert it into adipic acid, the foreign gene is as follows: (i) DNA encoding a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28, 29 or 30, (ii) DNA encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added with respect to the amino acid sequence shown in SEQ ID NO: 28, 29 or 30, (iii) DNA consisting of a polynucleotide sequence having 80% or more sequence identity with the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, (iv) DNA encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added with respect to the amino acid sequence of the protein encoded by the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, (v) DNA that hybridizes under stringent conditions with DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, or (vi) DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60 is a recombinant microorganism.

2. The foreign gene is as follows: (vii) DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 28, 29 or 30, or (viii) DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60 is the recombinant microorganism according to Claim 1.

3. The foreign gene is derived from at least one selected from Lachnospiraceae bacterium, Clostridium akagii and Treponema saccharophilum, and is the recombinant microorganism according to Claim 1 or 2.

4. The recombinant microorganism has a production pathway for C6 compounds, and the C6 compound is at least one selected from the group consisting of adipic acid and 2-hexenedioic acid, and is the recombinant microorganism according to Claim 1 or 2.

5. The recombinant microorganism according to claim 1 or 2, belonging to the genus Escherichia, Bacillus, Corynebacterium, Arthrobacter, Brevibacterium, Clostridium, Zymomonas, Pseudomonas, Burkholderia, Streptomyces, Rhodococcus, Synechocystis, Alkalihalobacillus, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, or Aspergillus.

6. The recombinant microorganism according to claim 1 or 2, wherein the recombinant microorganism is Escherichia coli.

7. The recombinant microorganism according to claim 1 or 2, further comprising at least one selected from the group consisting of a gene encoding 2-enoyl reductase, a gene encoding 2,4-dienoyl-CoA reductase, a gene encoding FAD-dependent oxidoreductase, and a gene encoding NADH-flavin oxidoreductase / NADH oxidase.

8. A method for producing a C6 compound using the recombinant microorganism according to claim 1 or 2, wherein the C6 compound is at least one selected from the group consisting of adipic acid and 2-hexenedioic acid.

9. having a reducing activity which is at least one of (a) an activity of reducing muconic acid to convert it into 2-hexenedioic acid and (b) an activity of reducing 2-hexenedioic acid to convert it into adipic acid, and (b) the following: (i) consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28, 29, or 30, (ii) consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added with respect to the amino acid sequence shown in SEQ ID NO: 28, 29 or 30, (iii) encoded by DNA consisting of a polynucleotide sequence having 80% or more sequence identity with the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, (iv) consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added with respect to the amino acid sequence of the protein encoded by the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, (v) encoded by DNA that hybridizes under stringent conditions with DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60, or (vi) a recombinant protein encoded by DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60.

10. (vii) consisting of the amino acid sequence shown in SEQ ID NO: 28, 29 or 30, or (viii) the recombinant protein according to claim 9, encoded by DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 58, 59 or 60.

11. A method for producing a C6 compound using the recombinant protein according to claim 9 or 10, The method for production, wherein the C6 compound is at least one selected from the group consisting of adipic acid and 2-hexenedioic acid.

Images