JP2024092581A - Recombinant halophilic microorganisms capable of producing carboxylic acids - Google Patents

Abstract

To provide a halophilic recombinant microbe capable of carboxylic acid production.SOLUTION: The present invention provides a halophilic recombinant microbe capable of carboxylic acid production, where the carboxylic acid is selected from C5 carboxylic acid, C6 carboxylic acid and aromatic carboxylic acid.SELECTED DRAWING: None

Description

The present invention relates to a recombinant halophilic microorganism capable of producing carboxylic acids, which are industrially useful compounds, and a method for producing carboxylic acids using the recombinant microorganism.

Adipic acid (CAS No. 124-04-9) is an industrially important monomer compound used as a raw material for polyamides such as nylon 66, urethanes, and plasticizers.

Adipic acid is currently generally produced by chemical synthesis, for example by oxidizing cyclohexanol alone or a mixture of cyclohexanol and cyclohexanone (K/A oil) with nitric acid.

In recent years, there are concerns that fossil fuels will be depleted and are considered to be a factor in global warming, so in chemical production processes, there is a desire to shift from fossil fuel-derived raw materials to renewable raw materials, such as biomass-derived raw materials, and production methods using fermentation production by microorganisms whose metabolism has been modified by genetic engineering have been proposed. For example, the biosynthesis of adipic acid, 6-aminocaproic acid, and caprolactam by microorganisms has been reported (Patent Document 1).

Adipic acid can also be produced from any of the three isomers of muconic acid, namely cis,cis-muconic acid; cis,trans-muconic acid; and trans,trans-muconic acid, by chemical hydrogenation. For example, it has been reported that adipic acid can be produced from renewable biomass feedstocks by hydrogenation after biosynthesis of cis,cis-muconic acid through fermentation by a microorganism whose metabolism has been modified by genetic engineering (Patent Document 2).

On the other hand, in the fermentative production of dicarboxylic acids such as succinic acid by microorganisms, it is necessary to add ammonia and/or sodium hydroxide in an amount equal to or greater than the amount of dicarboxylic acid to be synthesized in order to adjust the pH. Adding a large amount of pH adjuster significantly reduces the growth of the microorganisms and the productivity of the target compound. There are reports that various pH adjusters have been investigated in the fermentative production of succinic acid and have improved productivity (Non-Patent Document 1), but the decrease in productivity associated with the accumulation of succinate salts has not been fundamentally resolved.

In order to produce dicarboxylic acids by fermentation while maintaining high productivity using microorganisms, it is considered desirable to use, as a production host, a microorganism that is salt-tolerant or halophilic and can grow in an environment with a high dicarboxylate concentration. For example, it has been reported that it is possible to accumulate polyhydroxybutyrate (PHB), a raw material for biodegradable polymers, from basic waste glycerol by utilizing the tolerance of halophilic microorganisms to high salt concentration environments (Non-Patent Document 2).

However, unlike when the general-purpose Escherichia coli is used as a production host, no technology has been established to increase the production of target proteins in halophilic microorganisms. As a result, no halophilic microorganisms that produce carboxylic acids have been reported to date.

Patent No. 5951990 Patent No. 5487987

American Institute of Chemical Engineers Biotechnol. Prog., 25: 116-123, 2009 Kawata and Aiba, Biosci. Biotechnol. Biochem., 74, 175-177, 2010

In view of the above-mentioned current situation, the objective of the present invention is to provide a halophilic recombinant microorganism capable of producing carboxylic acids.

In order to solve the above problems, the inventors conducted extensive research and discovered that it is possible to impart the ability to produce the desired carboxylic acid by introducing an exogenous gene into a halophilic host microorganism.

That is, the present invention provides:
[1] A recombinant halophilic microorganism capable of producing a carboxylic acid, comprising:
a recombinant microorganism, wherein the carboxylic acid is selected from a C5 carboxylic acid, a C6 carboxylic acid, and an aromatic carboxylic acid;
[2] The recombinant microorganism according to [1], wherein the carboxylic acid is at least one selected from levulinic acid, adipic acid, 3-oxoadipic acid, 3-hydroxyadipic acid, and 2,3-dehydroadipic acid;
[3] The recombinant microorganism according to [1] or [2], wherein the recombinant microorganism expresses at least one protein selected from the group consisting of β-ketoadipyl-CoA thiolase (EC: 2.3.1.174), 3-oxoadipyl-CoA dehydrogenase (EC: 1.1.1.157), 3-hydroxyadipyl-CoA hydratase (EC: 4.2.1.17), 2,3-dehydroadipyl-CoA dehydrogenase (EC: 1.3.99.-), and acyl-CoA thioesterase (EC: 3.1.2.-);
[4] The recombinant microorganism according to [3], wherein an upstream sequence selected from a Pslp promoter, a Pald promoter, and a Psod promoter is arranged upstream of the base sequence encoding the protein;
[5] The recombinant microorganism according to [3], wherein rare codons are removed from a base sequence encoding the protein.
[6] The recombinant microorganism according to [1], wherein the carboxylic acid is at least one selected from protocatechuic acid and cis,cis-muconic acid;
[7] The recombinant microorganism according to [6], wherein the recombinant microorganism expresses at least one protein selected from the group consisting of 3-dehydroshikimate dehydratase (EC: 4.2.1.118), protocatechuate decarboxylase (EC: 4.1.1.63), and catechol 1,2-dioxygenase (EC: 1.13.11.1);
[8] The recombinant microorganism according to [7], further expressing enolate reductase (EC: 1.3.1.31);
[9] The recombinant microorganism according to [8], wherein a base sequence of a promoter selected from a Pslp promoter, a Pald promoter, and a Psod promoter is arranged upstream of a base sequence encoding the protein:
[10] The recombinant microorganism according to [2] or [7], wherein the microorganism is of the genus Bacillus;
[11] The recombinant microorganism according to [10], wherein the microorganism is Bacillus pseudofirmus, Bacillus halodurans or Bacillus marmarensis;
[12] The recombinant microorganism according to [11], wherein the microorganism is Bacillus pseudofirmus;
[13] A method for producing a carboxylic acid using the recombinant microorganism according to [1];
[14] The method according to [13], wherein the carboxylic acid is at least one selected from the group consisting of levulinic acid, adipic acid, 3-oxoadipic acid, 3-hydroxyadipic acid, and 2,3-dehydroadipic acid;
[15] The method according to [13], wherein the carboxylic acid is at least one selected from the group consisting of protocatechuic acid and cis,cis-muconic acid;
[16] The method according to [15], which comprises producing cis,cis-muconic acid using the recombinant microorganism according to [1] and converting the produced muconic acid into adipic acid by hydrogenation;
[17] The production method according to [13], comprising a culture step of culturing the recombinant microorganism in a medium containing 5% by mass or more of an inorganic salt to obtain a culture solution containing a carboxylic acid;
[18] A culturing step of culturing the recombinant microorganism to obtain a culture solution containing bacterial cells;
and a reaction step of contacting the culture solution and/or the bacterial cells with an aqueous solution containing 5% by mass or more of an inorganic salt to obtain a reaction solution containing a carboxylic acid;
[19] The production method according to [18], further comprising a removal step of removing the recombinant microorganism from the culture solution or reaction solution.
[20] The production method according to [18], which comprises a concentration step of concentrating the culture solution or reaction solution.
[21] The method according to [17], wherein the inorganic salt is sodium carbonate and/or sodium sulfate;
[22] The method according to [13], wherein the carboxylic acid is produced by fermentation of at least one of sugar, carbon dioxide, synthetic gas, methanol, and amino acids by the recombinant microorganism;
[23] The method according to [13], further comprising a purification step of purifying the obtained carboxylic acid.

The present invention provides a halophilic microorganism capable of producing carboxylic acids.

FIG. 1A is a schematic diagram showing an example of a synthetic pathway for a carboxylic acid. FIG. 1B is a schematic diagram showing an example of a synthetic pathway for a carboxylic acid. FIG. 2 shows the amino acid sequence and base sequence of each enzyme. FIG. 3 shows the amino acid sequence and base sequence of each enzyme. FIG. 4 shows the base sequences of SEQ ID NOs: 17 to 19 synthesized in the examples. FIG. 5 shows the nucleotide sequences of the primers used in the examples. FIG. 6 shows the base sequences of pAKAD08 to pAKAD16 constructed in the Examples. FIG. 7 shows gene expression levels. FIG. 8 shows the base sequences of the primers used in the examples. FIG. 9 shows the base sequence of the promoter used in the examples. FIG. 10 shows protein activity. FIG. 11 shows the base sequences of the AKAD-401 and AKAD-402 strains prepared in the Examples. FIG. 12 shows the base sequences of the primers used in the examples.

Hereinafter, the embodiment for carrying out the present invention (hereinafter also referred to as "the present embodiment") will be described in detail. Note that the present invention is not limited to the present embodiment, and various modifications can be made within the scope of the gist of the present invention. Furthermore, unless otherwise specified, the acquisition of DNA described in this specification, the preparation of vectors, and the genetic manipulation such as transformation can be performed by the 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 Genetic Engineering Experiment Notes (Yodosha, Takaaki Tamura). Unless otherwise specified in this specification, the nucleotide sequence is described from the 5' to the 3' direction. In this specification, the terms "polypeptide" and "protein" are used interchangeably. The term "genetically modified microorganism" is also referred to simply as "recombinant microorganism."

In this specification, a numerical range indicated using "~" indicates a range that includes the numerical values before and after "~" as the minimum and maximum values, respectively. In numerical ranges described in stages in this specification, the upper limit or lower limit of a numerical range in one stage can be arbitrarily combined with the upper limit or lower limit of a numerical range in another stage.

As used herein, the terms "endogenous" or "endogenous" are used to mean that the unmodified host microorganism possesses the referenced gene or the protein (typically an enzyme) encoded thereby, regardless of whether the gene is functionally expressed to the extent that it is capable of carrying out a dominant biochemical reaction within the host cell.

The terms "foreign" or "exogenous" are used herein to refer to the introduction of a gene or nucleic acid sequence according to the present invention into a host when the host microorganism does not have the gene to be introduced according to the present invention before genetic recombination, does not substantially express the enzyme from that gene, and has the amino acid sequence of the enzyme encoded by a different gene but does not express the comparable endogenous enzyme activity after genetic recombination. The terms "foreign" and "exogenous" are used interchangeably herein.

The recombinant microorganism of the present invention is a halophilic recombinant microorganism capable of producing carboxylic acid. Specifically, a halophilic host microorganism has been modified to have carboxylic acid production ability. The recombinant microorganism of the present invention is a microorganism suitable for producing high concentrations of carboxylate salts because it has salt tolerance.

The recombinant microorganism of the present invention has the ability to produce a carboxylic acid. In relation to the microorganism of the present invention, "having the ability to produce a carboxylic acid" refers to a microorganism that produces a carboxylic acid at any stage of the process of carboxylic acid production using the microorganism. Specifically, the medium obtained by culturing the microorganism may contain a carboxylic acid, or a carboxylic acid may be produced by adding a precursor of the carboxylic acid, for example, protocatechuic acid and/or catechol, to the medium and culturing a microorganism that converts the carboxylic acid precursor into a carboxylic acid. "Microorganisms having the ability to produce a carboxylic acid" include microorganisms that have one or more of these properties.

In the present specification, the expression "having a carboxylic acid producing ability" in relation to a recombinant microorganism means that the microorganism has a carboxylic acid production pathway. In relation to a compound, the expression "having a production pathway" in relation to the present invention means that the recombinant microorganism of the present invention expresses sufficient amounts of enzymes for each reaction step in the production pathway of the compound to proceed, and is capable of biosynthesizing the compound. In other words, the recombinant microorganism of the present invention expresses sufficient amounts of enzymes for each reaction step in the production pathway of the carboxylic acid to proceed, and is capable of biosynthesizing the carboxylic acid. The recombinant microorganism of the present invention may be a host microorganism that originally has the ability to produce a carboxylic acid, or may be a host microorganism that does not originally have the ability to produce the compound, but has been modified to have the ability to produce a carboxylic acid.

In the present invention, halophilic microorganisms that can be used as hosts are a general term for microorganisms that can cope with high salt stress. Microorganisms are classified according to the optimum salt concentration for growth into non-halophilic bacteria, whose optimum salt concentration for growth is 0 to less than 0.2 M sodium chloride, mild halophilic bacteria, whose optimum salt concentration for growth is 0.2 M to less than 0.5 M sodium chloride, moderate halophilic bacteria, whose optimum salt concentration for growth is 0.5 M to less than 2.5 M sodium chloride, and severe halophilic bacteria, whose optimum salt concentration for growth is 2.5 M to less than 5.2 M sodium chloride. All of these except non-halophilic bacteria are halophilic microorganisms according to the present invention. Here, halophilic bacteria whose optimum salt concentration for growth is 0.2 M sodium chloride or more are considered to be halotolerant, and "halotolerant" microorganisms include "halophilic" microorganisms.

The inventors believed that if a naturally occurring halophilic microorganism capable of growing in an environment with a sodium chloride concentration of 0.2 M or more could be used for the production of carboxylic acids, it would be possible to avoid the decline in productivity associated with the accumulation of carboxylates in the medium. As a result of extensive investigations, they constructed a recombinant microorganism modified to have carboxylic acid production ability, using a halophilic microorganism capable of growing in the presence of a high concentration of adipate, 100 g/L, as a host. Note that there have been no previous examples of applying halophilic microorganisms to carboxylic acid production.

In other words, the inventors have succeeded in imparting carboxylic acid production ability to a halophilic microorganism, and have found that by using such a microorganism, even if carboxylates accumulate in the medium, the decrease in productivity associated with the accumulation can be avoided.

The host microorganism includes various halophilic microorganisms, non-limiting examples of which include bacteria of the genus Bacillus, Halomonas, Halobacteroides, and Salinibacter. The host microorganism is preferably a bacterium of the genus Bacillus, specific examples of which include Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis. Among these, it is particularly preferable to use Bacillus pseudofirmus.

In this specification, "carboxylic acid" includes monocarboxylic acids as well as carboxylic acids having multiple carboxyl groups such as dicarboxylic acids. Examples of carboxylic acids include C5 carboxylic acids, C6 carboxylic acids, and aromatic carboxylic acids. Examples of C5 carboxylic acids include levulinic acid, examples of C6 carboxylic acids include adipic acid, 3-oxoadipic acid, 3-hydroxyadipic acid, 2,3-dehydroadipic acid, and cis,cis-muconic acid, and examples of aromatic carboxylic acids include protocatechuic acid, but are not limited to these.

The following two pathways are examples of carboxylic acid biosynthesis pathways using the recombinant microorganism of the present invention (Figures 1A and 1B).

<Route 1>
In pathway 1, acetyl-CoA and succinyl-CoA produced in a metabolic pathway originally possessed by the host microorganism and using glucose as a starting material are converted to 3-oxoadipyl-CoA, and a carboxylic acid is biosynthesized from 3-oxoadipyl-CoA. The carboxylic acid biosynthesized in this pathway is at least one selected from levulinic acid, adipic acid, 3-oxoadipic acid, 3-hydroxyadipic acid, and 2,3-dehydroadipic acid. An example in which the carboxylic acid is adipic acid will be described below with reference to FIG. 1A.

In one aspect related to this pathway, a host microorganism is modified to express at least one protein selected from the group consisting of β-ketoadipyl CoA thiolase (EC: 2.3.1.174), 3-oxoadipyl CoA dehydrogenase (EC: 1.1.1.157), 3-hydroxyadipyl CoA hydratase (EC: 4.2.1.17), 2,3-dehydroadipyl CoA dehydrogenase (EC: 1.3.99.-), and acyl CoA thioesterase (EC: 3.1.2.-). Specifically, one or more of β-ketoadipyl CoA thiolase, 3-oxoadipyl CoA dehydrogenase, 3-hydroxyadipyl CoA hydratase, 2,3-dehydroadipyl CoA dehydrogenase, and acyl CoA thioesterase are introduced into the cells of the host microorganism. By carrying out such modifications, a carboxylic acid biosynthetic pathway is constructed. Preferably, the enzyme gene sequence is inserted into the genome sequence of the host microorganism.

In the example shown in Figure 1A, acetyl-CoA and succinyl-CoA, which are produced in the metabolic pathway inherent to the host microorganism, are condensed to produce 3-oxoadipyl-CoA (Figure 1A, step 1). This conversion is catalyzed, for example, by β-ketoadipyl-CoA thiolase (EC: 2.3.1.174). A representative gene sequence for this enzyme is PaaJ of Escherichia coli. The amino acid sequence of the E. coli PaaJ enzyme is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2 (Figure 2).

Next, in step 2, 3-oxoadipyl-CoA is reduced to produce 3-hydroxyadipyl-CoA. This conversion is catalyzed, for example, by 3-oxoadipyl-CoA dehydrogenase (EC: 1.1.1.157). A representative gene sequence for this enzyme is PaaH from Escherichia coli. The amino acid sequence of the E. coli PaaH enzyme is shown in SEQ ID NO: 3, and the base sequence is shown in SEQ ID NO: 4 (Figure 2).

In step 3, 3-hydroxyadipyl-CoA is dehydrated to produce 2,3-dehydroadipyl-CoA. This conversion is catalyzed, for example, by 3-hydroxyadipyl-CoA hydratase (EC: 4.2.1.17). A representative gene sequence for this enzyme is PaaF of Escherichia coli. The amino acid sequence of the E. coli PaaF enzyme is shown in SEQ ID NO: 5, and the base sequence is shown in SEQ ID NO: 6 (Figure 2).

In step 4, 2,3-dehydroxyadipyl-CoA is reduced to produce adipyl-CoA. This conversion is catalyzed, for example, by 2,3-dehydroadipyl-CoA dehydrogenase (EC: 1.3.99.-). A representative gene sequence for this enzyme is Ter from Thermobifida fusca. The amino acid sequence of the Ter enzyme is shown in SEQ ID NO: 7, and the base sequence is shown in SEQ ID NO: 8 (Figure 2).

In step 5, adipyl-CoA is hydrolyzed to produce adipic acid. This conversion is catalyzed, for example, by acyl-CoA thioesterase (EC: 3.1.2.-). A representative gene sequence for this enzyme is TesB from Escherichia coli. The amino acid sequence of the TesB enzyme is shown in SEQ ID NO: 9, and the base sequence is shown in SEQ ID NO: 10 (Figure 2).

In the pathway shown in Figure 1A, levulinic acid is produced as a by-product. Specifically, 3-oxoadipyl CoA is converted to levulinic acid via 3-oxoadipate. It is predicted that acyl-CoA thioesterase hydrolyzes 3-oxoadipyl CoA to 3-oxoadipate, and decarboxylase decarboxylates 3-oxoadipate to produce levulinic acid. The aforementioned reactions are catalyzed by any endogenous decarboxylase present in the host microorganism.

As described above, by introducing five exogenous genes into a host microorganism, the host microorganism can be endowed with the ability to produce a carboxylic acid (adipic acid in Figure 1A) from acetyl-CoA and succinyl-CoA produced in the metabolic pathway of the microorganism.

In producing the genetically modified microorganism of this embodiment, for example, a technique for introducing a foreign gene into the cells of a halophilic microorganism, a technique for introducing an arbitrary foreign gene sequence into a genome sequence, or a technique for removing an unnecessary gene sequence from a genome sequence can be used.

That is, the following techniques are included:
(1) A technology that combines a promoter that can be expressed in halophilic microorganisms with a gene involved in carboxylic acid production,
(1-1) The above-mentioned technique (1), which uses a sequence of an upstream region of a gene encoding an S-Layer protein (also referred to as "Slp"), a gene encoding an alanine dehydrogenase (also referred to as "Ald"), or a gene encoding a superoxide dismutase (also referred to as "Sod"), as a promoter to be used in the technique;
(1-2) The above-mentioned technique (1), in which rare codons with low codon frequencies in all genes of a host microorganism are removed from the base sequence encoding the gene involved in carboxylic acid production, which is the subject of the previous technique;
(2) The technique according to (1) above, characterized in that the technique uses a plasmid vector that is stably replicated in the cells of a halophilic microorganism;
(2-1) The technique according to (1) above, wherein the plasmid vector to be used in the technique is pUB110;
(3) The technique according to (1) above, characterized in that a desired trait is stably expressed in the cells of a halophilic microorganism.
(4) A technique for selectively modifying gene sequences on chromosomes by combining temperature-sensitive plasmid vectors and negative selection.
(5) The technique according to (4) above, characterized in that the technique stops replication of a plasmid carried by a microorganism or reduces the copy number of the plasmid carried by the microorganism at a temperature of 37° C. or higher.
(6) The technique according to (4) above, characterized in that the growth of a microorganism having a specific gene is inhibited in the presence of substances such as sucrose and 4-chlorophenylalanine, thereby selecting a microorganism not having the specific gene (negative selection);
(6-1) The technique according to (4) above, wherein the specific gene to be targeted by the technique is the levansucrase gene (sacB gene);
(6-2) The technique according to (4) above, wherein the specific gene to be the subject of the technique is a phenylalanine tRNA synthetase α subunit gene (pheS gene) derived from a host into which a mutation has been introduced into the base sequence.

In the present invention, a high expression promoter and a foreign gene may be carried in a plasmid vector and introduced into the cells of a halophilic microorganism to stably and highly express the foreign gene. In addition, a technique that combines a temperature-sensitive plasmid vector with negative selection allows the genome sequence carried by the halophilic microorganism to be arbitrarily edited. The cell line into which the gene has been introduced or the genome sequence has been edited using this technique may be a wild type in the usual sense, or may be an auxotrophic mutant or an antibiotic-resistant mutant. Furthermore, the cell line that can be used as the host cell of the present invention may already be transformed to have various marker genes related to the above-mentioned mutations. These techniques can provide properties that are useful for the production, maintenance and/or management of the recombinant microorganism of the present invention.

In an embodiment of Pathway 1 of the present invention, in a recombinant microorganism, a promoter base sequence may be placed upstream of the base sequence encoding the protein (enzyme). In one aspect, two or more promoters may be selected and placed upstream of each gene of multiple proteins, or one promoter may be placed upstream of a series of base sequences including genes of multiple proteins.

In this embodiment, in the recombinant microorganism, it is preferable that an upstream sequence of a gene encoding an S-Layer protein (also referred to as "Slp"), a gene encoding an alanine dehydrogenase (also referred to as "Ald"), or a gene encoding a superoxide dismutase (also referred to as "Sod") is arranged as a promoter upstream of a base sequence encoding the protein (enzyme) (above (1-1)). Specifically, the following is arranged upstream of the base sequence encoding the protein (enzyme):
- Pslp promoter,
- the Pald promoter, and
- It is preferable that the base sequence of a promoter selected from the Psod promoter is arranged. Examples of the sequences of each promoter are shown in SEQ ID NOs: 104 to 106 (Figure 9). In this way, by arranging the promoter sequence upstream of the gene encoding the target protein, the transcription efficiency of the gene encoding the target protein can be improved.

In the recombinant microorganism of the present invention, codon optimization may be further performed. By performing codon optimization, the efficiency of translation into the target protein (enzyme) can be improved. Codon optimization of a gene sequence is an operation to remove infrequently used codons. Thus, in one embodiment, rare codons are removed from the base sequence encoding the protein (enzyme) in the recombinant composition. Preferably, it is an operation to change infrequently used codons to frequently used codons, and more preferably, it is an operation to change the gene sequence to one composed only of the most frequently used codons.

<Route 2>
In this pathway, 3-dehydroshikimic acid (DHS), which is produced in a metabolic pathway that uses glucose as a starting material and is inherent to the host microorganism, is converted to cis,cis-muconic acid via protocatechuic acid and catechol (FIG. 1B). The carboxylic acid biosynthesized in this pathway is at least one selected from protocatechuic acid and cis,cis-muconic acid.

In one aspect related to this pathway, one or more of 3-dehydroshikimate dehydratase (EC: 4.2.1.118), protocatechuate decarboxylase (EC: 4.1.1.63), and catechol 1,2-dioxygenase (EC: 1.13.11.1) are introduced into the cells of a host microorganism. By carrying out such modifications, a carboxylic acid biosynthetic pathway is constructed. Preferably, the enzyme gene sequence is inserted into the genome sequence of the host microorganism.

In the example shown in Figure 1B, 3-dehydroshikimic acid (DHS) produced in the metabolic pathway inherent to the host microorganism is converted to 3,4-dihydroxybenzoic acid (protocatechuic acid) by dehydration (Figure 1B, step 1). This conversion is catalyzed, for example, by 3-dehydroshikimic acid dehydratase. A representative gene sequence for this enzyme is QuiC from Acinetobacter Baylyi. The amino acid sequence of the QuiC enzyme is shown in SEQ ID NO: 11, and the nucleotide sequence is shown in SEQ ID NO: 12 (Figure 3).

In step 2, 3,4-dihydroxybenzoic acid is decarboxylated to produce 1,2-benzenediol (catechol). This conversion is catalyzed, for example, by protocatechuate decarboxylase (EC: 4.1.1.63). A representative gene sequence for this enzyme is UbiD from Klebsiella pneumoniae. The amino acid sequence of the UbiD enzyme is shown in SEQ ID NO: 13, and the base sequence is shown in SEQ ID NO: 14 (Figure 3).

In step 3, 1,2-benzenediol is oxidized to produce cis,cis-muconic acid. This conversion is catalyzed, for example, by catechol 1,2-dioxygenase (EC: 1.13.11.1). A representative gene sequence for this enzyme is CatA from Acinetobacter Baylyi. The amino acid sequence of the CatA enzyme is shown in SEQ ID NO: 15, and the base sequence is shown in SEQ ID NO: 16 (Figure 3).

As described above, by introducing three exogenous genes into a host microorganism, the host microorganism can be endowed with the ability to produce a carboxylic acid (protocatechuic acid and cis, cis-muconic acid in FIG. 1B) from 3-dehydroshikimic acid produced in the metabolic pathway of the microorganism. The carboxylic acid produced here may be subjected to further reaction and converted into another carboxylic acid. For example, cis, cis-muconic acid may be further subjected to hydrogenation and converted into adipic acid. In the hydrogenation, cis, cis-muconic acid is converted into adipic acid via, for example, 2,3-dehydroadipic acid. The method of hydrogenating cis, cis-muconic acid is not particularly limited, and examples include hydrogenation using a metal catalyst and hydrogenation using an enzyme. In a preferred embodiment, hydrogenation of cis, cis-muconic acid is performed using a metal catalyst. In another preferred embodiment, cis,cis-muconic acid is hydrogenated by enolate reductase (EC: 1.3.1.31) and converted to adipic acid.

In producing a genetically modified microorganism according to an embodiment of Route 2, for example, a technique for introducing a foreign gene into the cells of a halophilic microorganism, a technique for introducing an arbitrary foreign gene sequence into a genome sequence, or a technique for removing an unnecessary gene sequence from a genome sequence can be used, and the techniques (1) to (6) described above for the embodiment of Route 1 can also be used in the embodiment of Route 2.

In an embodiment of pathway 2 of the present invention, in a recombinant microorganism, a promoter selected from the group of promoters described in Table A above is arranged as an upstream sequence upstream of a base sequence encoding the protein. In a recombinant microorganism, it is preferable that an upstream sequence of a gene encoding Slp, a gene encoding Ald, or a gene encoding Sod is arranged as a promoter upstream of a base sequence encoding the protein (enzyme) (above (1-1)), and specifically, upstream of a base sequence encoding the protein (enzyme):
- Pslp promoter,
- the Pald promoter, and
- It is preferable that the base sequence of a promoter selected from the Psod promoter is arranged. Examples of the sequences of each promoter are shown in SEQ ID NOs: 104 to 106 (Figure 9). In this way, by arranging the promoter sequence upstream of the gene encoding the target protein, the transcription efficiency of the gene encoding the target protein can be improved.

Furthermore, the recombinant microorganism of the present invention may be subjected to codon optimization. By performing codon optimization, the efficiency of translation into the target protein (enzyme) can be improved. Codon optimization of a gene sequence is an operation to remove infrequently used codons. Thus, in one embodiment, the recombinant composition has rare codons removed from the base sequence encoding the protein (enzyme). Preferably, it is an operation to change infrequently used codons to frequently used codons, and more preferably, it is an operation to change the gene sequence to one composed only of the most frequently used codons.

The exogenous proteins (i.e., enzymes) described for pathways 1 and 2 are (A), (B), (C), (D), and (E) below:
(A) introducing into a host microorganism an exogenous gene encoding the protein;
(B) increasing the copy number of the endogenous gene for said protein in a host microorganism;
(C) introducing a mutation into an expression regulatory region of an endogenous gene for said protein in a host microorganism;
The protein can be introduced and/or expressed by performing one or more genetic manipulations selected from the group consisting of: (D) a manipulation for replacing the expression regulatory region of an endogenous gene for the protein in a host microorganism with an exogenous regulatory region capable of high expression; and (E) a manipulation for deleting the regulatory region of an endogenous gene for the protein in a host microorganism.

The genetically modified organism obtained as described above is cultured and maintained under conditions suitable for its growth and/or maintenance for the production of carboxylic acids. Therefore, another aspect of the present invention relates to a method for producing carboxylic acids using the recombinant microorganism described above. The carboxylic acid is, for example, at least one selected from a C5 carboxylic acid, a C6 carboxylic acid, and an aromatic carboxylic acid, and is preferably at least one selected from the group consisting of levulinic acid, adipic acid, 3-oxoadipic acid, 3-hydroxyadipic acid, 2,3-dehydroadipic acid, protocatechuic acid, and cis,cis-muconic acid.

The manufacturing method of the present invention includes, for example, the following steps:

(Cultivation process)
In this step, the recombinant microorganism is cultured in a medium. In one embodiment, the recombinant microorganism is cultured in a medium containing an inorganic salt to obtain a culture solution containing a carboxylic acid. The concentration of the inorganic salt in the medium is, for example, 5 mass% or more. The inorganic salt contained in the medium is sodium carbonate and/or sodium sulfate. In another embodiment, the recombinant microorganism is cultured to obtain a culture solution containing the bacterial cells.

(Reaction step)
In this step, after obtaining a culture solution containing the bacterial cells in the culture step, the culture solution and/or the bacterial cells are contacted with an aqueous solution containing 5% by mass or more of an inorganic salt to obtain a reaction solution containing a carboxylic acid.

The production method of the present invention may further include a removal step of removing the recombinant microorganism from the culture solution or reaction solution obtained in the previous step, and/or a concentration step of concentrating the culture solution or reaction solution obtained in the previous step.

(Separation process)
This step may include a step of separating and/or purifying the target compound, carboxylic acid, from the culture solution or reaction solution obtained in the previous step. The separation and/or purification step may be performed by any method, such as, but not limited to, centrifugation, filtration, membrane separation, crystallization, extraction, distillation, adsorption, phase separation, ion exchange, and various types of chromatography. In the separation and/or purification step, one method may be selected, or multiple methods may be combined.

In the production method of the present invention, a person skilled in the art can easily determine suitable medium compositions, culture conditions, and culture times for transformants derived from various host microbial cells.

The medium may be a natural, semi-synthetic, or synthetic medium containing one or more carbon sources, nitrogen sources, inorganic salts, vitamins, and optionally trace elements or vitamins. However, it goes without saying that the medium used must adequately meet the nutritional requirements of the genetically modified organism to be cultured.

Carbon sources include D-glucose, sucrose, lactose, fructose, maltose, oligosaccharides, polysaccharides, starch, cellulose, rice bran, blackstrap molasses, fats and oils (e.g., soybean oil, sunflower oil, peanut oil, coconut oil, etc.), fatty acids (e.g., palmitic acid, linoleic acid, linolenic acid, etc.), alcohols (e.g., glycerol, ethanol, etc.), and organic acids (e.g., acetic acid, lactic acid, succinic acid, etc.). It may also be biomass containing D-glucose. Suitable biomass includes corn decomposition liquid and cellulose decomposition liquid. Other carbon sources include sugar, carbon dioxide, synthetic gas, methanol, and amino acids. That is, in one embodiment, in the production method according to the present invention, the carboxylic acid is produced by fermentation of at least one of sugar, carbon dioxide, synthetic gas, methanol, and amino acids by a recombinant microorganism. The above carbon sources can be used individually or as a mixture.

When biomass-derived raw materials are used, the product carboxylic acids can be clearly distinguished from synthetic raw materials derived from, for example, petroleum, natural gas, or coal by measuring the biobased carbon content based on Carbon-14 (radiocarbon) analysis as specified in ISO 16620-2 or ASTM D6866.

Nitrogen sources include nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat extract, malt extract, corn steep liquor, soy flour, and urea) or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, sodium nitrate, ammonium nitrate, etc.). These nitrogen sources can be used individually or as a mixture.

In addition, if the transformant expresses a useful additional trait, for example, if it has an antibiotic resistance marker, the medium may contain the corresponding antibiotic. This reduces the risk of contamination with unwanted bacteria during fermentation. Examples of antibiotics include, but are not limited to, ampicillin, kanamycin, chloramphenicol, tetracycline, erythromycin, streptomycin, and spectinomycin.

If the host microorganism cannot assimilate the above carbon sources such as cellulose and polysaccharides, the host microorganism can be adapted to carboxylic acid production using these carbon sources by using known genetic engineering techniques such as introducing foreign genes into the host microorganism. Examples of foreign genes include cellulase genes and amylase genes.

The culture may be batch or continuous. In either case, the carbon source or the like may be added at an appropriate time during the culture. Furthermore, the culture should be continued while maintaining an appropriate temperature, oxygen concentration, pH, etc. The appropriate culture temperature for transformants derived from general microbial host cells is usually in the range of 15°C to 50°C, preferably 25°C to 37°C. When the host microorganism is aerobic, shaking (flask culture, etc.) or stirring/aeration (jar fermenter culture, etc.) is required to ensure an appropriate oxygen concentration during fermentation. Those culture conditions can be easily determined by those skilled in the art.

According to the present invention, a recombinant halophilic microorganism capable of producing carboxylic acids can be provided. Since the recombinant microorganism is a modified version of a halophilic host microorganism, even if carboxylates produced by the microorganism accumulate in the medium and the salt concentration increases, a decrease in productivity due to salt accumulation can be avoided. In addition, a novel carboxylic acid biosynthesis pathway can be provided that utilizes the glucose metabolic pathway inherent to the host microorganism.

The present invention will be described below based on examples, but the present invention is not limited to these examples.

All PCRs shown in this example were performed using PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio), RNA extraction was performed using Qiagen (product name, manufactured by Qiagen), RNA reverse transcription was performed using PrimeScript RT reagent Kit with gDNA Eraser (product name, manufactured by Takara Bio), and quantitative PCR was performed using TB Green Premix Ex Taq (product name, manufactured by Takara Bio).

Electroporation was used to transform Bacillus pseudofirmus. In the electroporation method, a 0.1 cm wide cuvette containing 1 μl of plasmid DNA together with 60 μl of competent cells was attached to a gene pulsar (Bio-Rad), and a pulse of 2.5 kV voltage, 200 Ω resistance, and 25 μF capacitance was applied to the cuvette. After 3 hours of recovery culture at 30°C, the cells were spread on 181 medium containing 10 μg/mL chloramphenicol to obtain transformants. The composition of 181 medium is shown in Table 1.

Example 1: Construction of a halophilic carboxylic acid producing bacterium (Example 1-a) Preparation of a chromosomal insertion plasmid Bacillus pseudophyllum OF4 strain (JCM17055 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project) was cultured in 181 medium (2 ml) with shaking at 37°C. After the culture was completed, the cells were collected from the culture liquid, and genomic DNA was extracted using Nucleo Spin Tissue (product name, manufactured by MACHEREY-NAGEL). A homology region including the promoter region was PCR amplified (fragment 1) using a primer set labeled A and B (Figure 5). The reaction conditions were 98°C (10 sec), 55°C (5 sec), 72°C (30 sec), and 30 cycles.

Plasmid pE197CTPK (internationally deposited as NITE BP-03287 at the National Institute of Technology and Evaluation, National Patent Microorganism Depository (NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on September 16, 2020) was PCR amplified using a primer set marked with C and D (Figure 5) (fragment 2). The reaction conditions were 98°C (10 sec), 55°C (5 sec), 72°C (30 sec), 30 cycles. Fragment 2 was used as the vector side, and fragment 1 was ligated as an insert to construct pAKAD01-03.

(Example 1-b) Cloning of carboxylic acid synthesis genes Genes with sequences shown in SEQ ID NOs: 17 to 19 were synthesized (FIG. 4). Using the synthetic genes as a template, PCR amplification was performed with a primer set with E and F (FIG. 5) (fragment 3). The reaction conditions were 98°C (10 sec), 55°C (5 sec), 72°C (30 sec), 30 cycles. Using the above chromosome insertion plasmid as a template, PCR amplification was performed with a primer set with G and H (FIG. 5) (fragment 4). Fragment 4 was used as the vector side, and fragment 3 was ligated as an insert to construct pAKAD04 to 06. That is, plasmids pAKAD04 to 06 were constructed by incorporating SEQ ID NOs: 17 to 19 prepared by artificial gene synthesis into vector plasmids (pAKAD01 to 03), respectively.

(Example 1-c) Obtaining a transformant The pAKAD04 constructed in Example 1-b was transformed into the Bacillus pseudophyllum OF4 strain to obtain the AKADp-101 strain.

(Example 1-d) Obtaining a plasmid-inserted strain The AKADp-101 strain obtained in Example 1-c was cultured in 181 medium containing 10 μg/mL chloramphenicol at 30° C. for 1 day, then diluted 100-fold and spread onto 181 medium containing 10 μg/mL chloramphenicol and cultured at 43° C. to obtain a chromosomal insertion strain AKADp-201 strain in which the plasmid had been homologously recombined into the chromosome.

(Example 1-e) Obtaining a gene-inserted strain (1)
The AKADp-201 strain obtained in Example 1-d was cultured for 1 day in 181 medium containing 10 μg/mL chloramphenicol at 30 ° C., then diluted 100-fold and spread on 181 medium containing 5 mM 4-chlorophenylalanine, and cultured at 30 ° C. to obtain a gene insertion strain AKAD-001 strain in which the plasmid and each gene region were removed from the chromosome and an external gene was inserted. The removal of the plasmid and the insertion of the gene were confirmed by PCR using a primer set with I and K (Figure 5). The reaction conditions were 98 ° C. (10 sec), 55 ° C. (5 sec), 72 ° C. (30 sec), and 30 cycles.

(Example 1-f) Acquisition of gene insertion strain (2)
Using pAKAD05 constructed in Example 1-b and the AKAD-001 strain obtained in Example 1-e, the same operations as in Examples 1-c to 1-e were performed to obtain a gene insertion strain, AKAD-002 strain, in which an external gene was inserted.

(Example 1-g) Acquisition of gene insertion strain (3)
Using pAKAD06 constructed in Example 1-b and the AKAD-002 strain obtained in Example 1-f, the same operations as in Examples 1-c to 1-e were performed to obtain a gene insertion strain, AKAD-003 strain, into which an external gene was inserted.

The genotypes of the obtained gene insertion strains are shown in Table 2. The base sequences of the primers used are shown in Figure 5 as SEQ ID NOs: 20 to 89.

Example 2: Evaluation of carboxylic acid production ability in the constructed strain (fermentor culture)
The AKAD-001 to AKAD-003 strains were cultured on a 181 medium plate at 37° C. for 1 day to form colonies. 2 mL of the 181 medium was placed in a 14 mL test tube, and a colony was inoculated from the plate using a platinum loop. The culture was carried out at 37° C. and 180 rpm until sufficient turbidity was obtained, and this was used as a preculture solution for main culture.

A BS jar medium (shown in Table 4) containing 40 g/L glucose was placed in a 100 mL jar culture device (model name: Bio Jr. 8, manufactured by Biot Co., Ltd.), 1 mL of preculture solution was added, and main culture was performed (cadaverine production test). The culture conditions were culture temperature: 37°C, culture pH: 7.5, alkali addition: 10% ammonia water, stirring speed: 750 rpm, and aeration speed: 0.1 vvm. Sampling was performed over time during the culture, and the turbidity of the bacterial cells in the culture solution and the carboxylic acid concentration in the culture supernatant were quantified. 8 ml of 50% glucose solution was added 24 hours and 48 hours after the start of culture.

The above culture solution was centrifuged at 10,000g for 3 minutes to collect the supernatant, and the carboxylic acid concentration of the culture supernatant was measured. Analysis was performed by GC/MS analysis using trimethylsilyl derivatization. 360 μL of an extract mixed with water:methanol:chloroform = 5:2:2 (v/v/v) was added to 40 μL of the culture solution centrifuged supernatant (added with disodium sebacate as an internal standard to a final concentration of 10 mM), and the mixture was thoroughly stirred with a vortex mixer. After centrifugation (16,000 × g, 5 minutes), 40 μL of the supernatant was collected in another microtube and centrifuged and dried for 1 hour with a centrifugal evaporator. 100 μL of methoxyamine hydrochloride 20 mg/mL pyridine solution was added to the obtained dried product, and the mixture was shaken at 30 ° C for 90 minutes. 50 μL of N-methyl-N-trimethylsilyl trifluoroacetamide was added, and the mixture was shaken at 37 ° C for 30 minutes. The reaction solution was used as a GC/MS measurement sample and analyzed under the following conditions.

GC/MS analysis conditions: Apparatus: GCMS-QP-2020NX (Shimadzu Corporation)
Column: Fused silica capillary tube, deactivated tube (length 1 m, outer diameter 0.35 mm, inner diameter 0.25 mm, manufactured by GL Sciences), InertCap 5MS/NP (length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm, manufactured by GL Sciences)
Sample injection volume: 1 μL
Sample introduction method: split (split ratio 25:1)
Vaporization chamber temperature: 230°C
Carrier gas: Helium Carrier gas linear velocity: 39.0 cm/sec Oven temperature: 80°C, held for 2 minutes → 15°C/min heating → 325°C, held for 13 minutes Ionization method: Electron ionization (EI)
Ionization energy: 70 eV
Ion source temperature: 230° C.
Scan range: m/z = 50 to 500

The bacterial cell density (OD), C6 dicarboxylic acid and levulinic acid concentrations (g/l) after 41 and 67.5 hours are shown in Table 4 (levulinic acid is a decomposition product of 3-oxoadipic acid). Levulinic acid and C6 dicarboxylic acid were not detected in the culture supernatant of AKAD-001. On the other hand, levulinic acid and 3-hydroxyadipic acid were detected in the culture supernatant of AKAD-002, and levulinic acid, 3-hydroxyadipic acid and adipic acid were detected in the culture supernatant of AKAD-003.

Example 3: Enhancement of carboxylic acid production pathway (Example 3-a) Preparation of gene expression plasmid Bacillus pseudophyllum OF4 strain was cultured at 37°C with shaking in 181 medium (2 ml). After the culture was completed, the cells were collected from the culture liquid, and genomic DNA was extracted using Nucleo Spin Tissue (product name, manufactured by MACHEREY-NAGEL). A homology region including the promoter region was PCR amplified (fragment 5) using a primer set labeled A and B. The reaction conditions were 98°C (10 sec), 55°C (5 sec), 72°C (30 sec), and 30 cycles.

Plasmid pAL351 (internationally deposited as NITE BP-02918 at the National Institute of Technology and Evaluation, National Patent Microorganism Depository (NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on March 18, 2019) was PCR amplified using a primer set labeled C and D (fragment 6). The reaction conditions were 98°C (10 sec), 55°C (5 sec), 72°C (30 sec), 30 cycles. Fragment 6 was used as the vector side, and fragment 5 was ligated as an insert to construct pAKAD07.

(Example 3-b) Cloning of enoyl-CoA dehydrogenase Genes (total of 9) having sequences shown in SEQ ID NOs: 90 to 98 were synthesized (FIG. 6). Using the above-mentioned synthetic gene and the gene synthesized in (Example 1-b) (SEQ ID NO: 18) as templates, PCR amplification was performed with a primer set with E and F (fragment 7). The reaction conditions were 98°C (10 sec), 55°C (5 sec), 72°C (30 sec), 30 cycles. Using the above gene expression plasmid as a template, PCR amplification was performed with a primer set with G and H (FIG. 5) (fragment 8). Fragment 8 was used as the vector side, and fragment 7 was ligated as an insert to construct pAKAD08-16.

(Example 3-c) Obtaining transformants The pAKAD08 to pAKAD16 constructed in Example 2-b were transformed into the AKAD-003 strain to obtain AKADp-301 to 309 strains.

(Example 3-d) Evaluation of C6 dicarboxylic acid productivity in the obtained strains AKADp-301 to 309 strains were cultured on a 181 medium plate containing 10 μg/mL chloramphenicol at 37° C. for 1 day to form colonies. 2 mL of 181 medium containing 10 μg/mL chloramphenicol was placed in a 14 mL test tube, and a colony was inoculated from the plate using a platinum loop. The culture was carried out at 37° C. and 180 rpm until sufficient turbidity was obtained, and this was used as a preculture solution for main culture.

A BS jar medium (shown in Table 5) containing 40 g/L glucose was placed in a 100 mL jar culture device (model name: Bio Jr. 8, manufactured by Biot Co., Ltd.), 1 mL of preculture solution was added, and main culture was performed (cadaverine production test). The culture conditions were culture temperature: 37°C, culture pH: 7.5, alkali addition: 10% ammonia water, stirring speed: 750 rpm, and aeration speed: 0.1 vvm. Sampling was performed over time during the culture, and the turbidity of the bacterial cells in the culture solution and the carboxylic acid concentration in the culture supernatant were quantified. 8 ml of 50% glucose solution was added 24 hours and 48 hours after the start of culture.

The above culture solution was centrifuged at 10,000 g for 3 minutes to collect the supernatant, and the concentrations of C6 dicarboxylic acids and levulinic acid in the culture supernatant were measured. Specifically, the carboxylic acid concentrations in the culture supernatant were quantified by GC-MS analysis.

Table 6 shows the cell density (OD), C6 dicarboxylic acid and levulinic acid concentrations (g/l) after 41, 48 and 67.5 hours (levulinic acid is a decomposition product of 3-oxoadipic acid). Adipic acid of 150 mg/L or more was detected in the culture supernatant of all AKADp-301 to 309 strains. In Example 2, adipic acid was detected at 6 mg/L in the culture supernatant of AKAD-003, which was not transformed with a plasmid, and the amount of adipic acid secreted by the transformants was significantly improved. In Example 2, the culture supernatant of AKAD-003 had a higher concentration of levulinic acid than adipic acid, but all transformants had a higher concentration of adipic acid than levulinic acid.

Example 4: Resistance to C6 compounds Escherichia coli K12 strain was cultured on an LB medium plate at 37° C. for 1 day to form colonies. 2 mL of LB medium was placed in a 14 mL test tube, and a colony was inoculated from the plate using a platinum loop. Culture was carried out at 37° C. and 180 rpm until sufficient turbidity was obtained, and this was used as a preculture solution for main culture.

Bacillus pseudophyllum OF4 strain was cultured on 181 medium plate at 37°C for 1 day to form colonies. 2 mL of 181 medium was placed in a 14 mL test tube, and a colony was inoculated from the plate using a platinum loop. The culture was carried out at 37°C and 180 rpm until sufficient turbidity was obtained, and this was used as a preculture solution for main culture.

BS medium (shown in Table 7) containing disodium adipate (ADA2Na) at concentrations of 0, 20, 40, 60, 80, and 100 g/L was added to a 2 mL deep well. 50 μL of preculture solution of Escherichia coli or Bacillus pseudophyllum OF4 strain was added and main culture was performed. Culture conditions were 37°C and 180 rpm. Sampling was performed after 43 hours, and the turbidity of the bacterial cells in the culture solution was quantified.

The growth rate at each concentration was calculated by setting the growth rate at 0 g/L of ADA2Na as 100 (Table 8). The growth rate of E. coli was halved at 20 g/L of ADA2Na, whereas the growth rate of Bacillus pseudophyllum OF4 did not change even at 100 g/L of ADA2Na.

Example 5: Selection of a high-expression promoter The same operations as in Example 1-c to Example 1-e were performed to insert a paaJ-paaH gene cassette with the following four types of base sequences added upstream into the chromosome of the Bacillus pseudofilamus OF4 strain. The four types of base sequences inserted are promoter sequences present upstream of the gapA gene, sod gene, ald gene, and slpA gene, respectively, and the respective strains were named AKAD-004 to 007 strains. The correspondence between each strain and the promoter used is shown in the table below (Table 9), and the promoter sequence is shown in FIG. 9. The four types of base sequences inserted were selected from sequences obtained by transcriptome analysis of the Bacillus pseudofilamus OF4 strain. Specifically, RNA was extracted when the Bacillus pseudofilamus OF4 strain was cultured in BS medium, and transcriptome analysis was performed. The above four types of promoter sequences were selected and used from the several thousand detected genes based on comparison of gene expression levels, etc.

The AKAD-004 to 007 strains were cultured on 181 medium plates at 37°C for 1 day to form colonies. 2 mL of 181 medium was placed in a 14 mL test tube, and a colony was inoculated from the plate using a platinum loop. The culture was carried out at 37°C and 180 rpm until sufficient turbidity was obtained, and this was used as a preculture solution for main culture.

2 ml of BS medium was added to a 14-mL test tube. 50 μL of precultured AKAD-004 to 007 strains were added and main culture was performed. Culture conditions were 37°C and 180 rpm. After 6 hours, the entire amount was collected and RNA was extracted. After reverse transcription of the extracted RNA, quantitative PCR was performed to measure the expression levels of the PpaaJ gene and the paaH gene (Figure 7). The primer list in Figure 8 was used for quantitative PCR, and the reaction conditions were 95°C (30 sec), {95°C (5 sec), 60°C (30 sec)} x 40 cycles, Melt curve.

For both the paaJ and paaH genes, the gene expression levels were highest when P_slpA was used as the promoter. The expression levels of the PpaaJ and PpaaH genes when P_gapA and P_slpA were used, respectively, differed by approximately 100-fold or more, demonstrating that P_slpA is a strong, high-expression promoter in the Bacillus pseudophyllum OF4 strain.

Example 6: Codon optimization (Example 6-a) Preparation of ddh gene expression plasmid The genes shown in SEQ ID NOs: 107 and 108 were synthesized. SEQ ID NO: 107 is the ddh gene of Corynebacteria glutamicum ATCC13032 strain, and SEQ ID NO: 108 is a base sequence obtained by optimizing SEQ ID NO: 107 with codons based on the codon usage frequency of Bacillus pseudofilamus OF4 strain. Specifically, as shown in Table 10, a codon with a low usage frequency was changed to a codon with a high usage frequency.

The synthetic gene described above was PCR amplified using the primer set in Table 13 marked with A and B (fragment 9). The reaction conditions were 98°C (10 sec), 55°C (5 sec), 72°C (30 sec), and 30 cycles. Using pAKAD07 prepared in Example 3-a as a template, PCR amplified using the primer set in Table 13 marked with C and D (fragment 10). The reaction conditions were 98°C (10 sec), 55°C (5 sec), 72°C (60 sec), and 30 cycles. Fragment 9 was used as the vector side, and fragment 10 was ligated as an insert to construct pAKAD17-18.

(Example 6-b) Preparation of ddh gene expression plasmid The constructed pAKAD17 to 18 were transformed into Bacillus pseudophyllum OF4 strain to obtain AKADp-401 to 402 strains. The sequences of the primers used are shown in FIG.

(Example 6-c) Comparison of Ddh activity AKADp-401 to 402 strains were cultured on a 181 medium plate containing 10 μg/mL chloramphenicol at 37 ° C. for 1 day to form colonies. 2 mL of 181 medium containing 10 μg/mL chloramphenicol was placed in a 14 mL test tube, and a colony was inoculated from the plate with a platinum loop, cultured at 37 ° C. and 180 rpm until sufficient turbidity was obtained, and this was used as a preculture solution for main culture.

2 ml of 181 medium containing 10 μg/mL chloramphenicol was added to a 14 mL test tube. 50 μL of the precultured AKADp-401-402 strains were added and main culture was performed. Culture conditions were 37°C and 180 rpm. After 6 hours, the cells were collected by centrifugation and disrupted by ultrasonic treatment on ice. The disrupted cell solution was incubated at 37°C for 1 hour with the composition shown in Table 11, and the wavelength at OD340 nm was measured over time using a multireader Infinite 200. The protein concentration in the disrupted cell solution was measured using a Bio-Rad Protein Assay Kit (product name, Bio-Rad).

Comparing Ddh activity with and without codon optimization, almost no activity was observed in the AKAD-401 strain, which has a base sequence that has not been codon optimized (SEQ ID NO: 107 (Figure 11)) (Figure 7). On the other hand, high activity was observed in the AKAD-402 strain, which has a base sequence that has been codon optimized (SEQ ID NO: 108 (Figure 11)) (Figure 10).

Claims (23)

A recombinant halophilic microorganism capable of producing carboxylic acids, comprising:
The recombinant microorganism, wherein the carboxylic acid is selected from a C5 carboxylic acid, a C6 carboxylic acid, and an aromatic carboxylic acid. The recombinant microorganism according to claim 1, wherein the carboxylic acid is at least one selected from levulinic acid, adipic acid, 3-oxoadipic acid, 3-hydroxyadipic acid, and 2,3-dehydroadipic acid. The recombinant microorganism according to claim 1 or 2, wherein the recombinant microorganism expresses at least one protein selected from the group consisting of β-ketoadipyl CoA thiolase (EC: 2.3.1.174), 3-oxoadipyl CoA dehydrogenase (EC: 1.1.1.157), 3-hydroxyadipyl CoA hydratase (EC: 4.2.1.17), 2,3-dehydroadipyl CoA dehydrogenase (EC: 1.3.99.-) and acyl CoA thioesterase (EC: 3.1.2.-). The recombinant microorganism according to claim 3, wherein an upstream sequence selected from a Pslp promoter, a Pald promoter, and a Psod promoter is arranged upstream of the base sequence encoding the protein. The recombinant microorganism according to claim 3, wherein rare codons have been removed from the base sequence encoding the protein. The recombinant microorganism according to claim 1, wherein the carboxylic acid is at least one selected from protocatechuic acid and cis,cis-muconic acid. The recombinant microorganism according to claim 6, wherein the recombinant microorganism expresses at least one protein selected from the group consisting of 3-dehydroshikimate dehydratase (EC: 4.2.1.118), protocatechuate decarboxylase (EC: 4.1.1.63) and catechol 1,2-dioxygenase (EC: 1.13.11.1). The recombinant microorganism of claim 7, further expressing enolate reductase (EC: 1.3.1.31). The recombinant microorganism according to claim 8, wherein a base sequence of a promoter selected from a Pslp promoter, a Pald promoter, and a Psod promoter is arranged upstream of the base sequence encoding the protein. The recombinant microorganism according to claim 2 or 7, wherein the microorganism is of the genus Bacillus. The recombinant microorganism according to claim 10, wherein the microorganism is Bacillus pseudofirmus, Bacillus halodurans or Bacillus marmarensis. The recombinant microorganism according to claim 11, wherein the microorganism is Bacillus pseudofirmus. A method for producing a carboxylic acid using the recombinant microorganism according to claim 1. The method according to claim 13, wherein the carboxylic acid is at least one selected from the group consisting of levulinic acid, adipic acid, 3-oxoadipic acid, 3-hydroxyadipic acid, and 2,3-dehydroadipic acid. The method according to claim 13, wherein the carboxylic acid is at least one selected from the group consisting of protocatechuic acid and cis,cis-muconic acid. The method according to claim 15, which uses the recombinant microorganism according to claim 1 to produce cis,cis-muconic acid and converts the produced muconic acid into adipic acid by hydrogenation. The method according to claim 13, further comprising a culture step of culturing the recombinant microorganism in a medium containing 5% by mass or more of inorganic salts to obtain a culture solution containing a carboxylic acid. A culturing step of culturing the recombinant microorganism to obtain a culture solution containing the bacterial cells;
The method according to claim 13, further comprising a reaction step of contacting the culture solution and/or the bacterial cells with an aqueous solution containing 5% by mass or more of an inorganic salt to obtain a reaction solution containing a carboxylic acid. The method according to claim 18, further comprising a removal step of removing the recombinant microorganism from the culture or reaction solution. The method according to claim 18, further comprising a concentration step of concentrating the culture solution or reaction solution. The method according to claim 17, wherein the inorganic salt is sodium carbonate and/or sodium sulfate. The method according to claim 13, wherein the carboxylic acid is produced by fermentation of at least one of sugar, carbon dioxide, synthetic gas, methanol, and amino acids by the recombinant microorganism. The method according to claim 13, further comprising a purification step of purifying the resulting carboxylic acid.