JP7645685B2 - Microorganism capable of producing 2-hydroxyisobutyric acid and method for producing 2-hydroxyisobutyric acid - Google Patents

Description

NPMD NITE P-03416

The present invention relates to a microorganism capable of producing 2-hydroxyisobutyric acid, and also to a method for producing 2-hydroxyisobutyric acid using the microorganism.

Methacrylic acid and its derivatives, as well as its polymers (hereinafter sometimes abbreviated as "methacrylic acid, etc.") are processed into acrylic sheets and used as molding materials for windows and electronic components, as well as for a variety of other applications such as paints and adhesives. Currently, methacrylic acid, etc. are mainly produced using petrochemical resources as raw materials, for example, the acetone ninhydrin method using acetone as the raw material or the direct oxidation method using isobutylene as the raw material. However, petrochemical resources are limited and the environmental impact is high, so there is a demand for the development of alternative methods.

Therefore, methods for producing methacrylic acid and the like using renewable resources instead of petrochemical resources have been reported. Such methods have attracted attention as methods that reduce the environmental burden and are highly cost-effective. For example, a method has been reported in which genetically engineered microorganisms are used to produce 2-hydroxyisobutyric acid (hereinafter sometimes abbreviated as "2-HIBA"), which can be easily chemically converted to methacrylic acid and the like (see, for example, Patent Documents 1 and 2).

Patent Document 1 reports that 2-HIBA is produced via acetoacetyl coenzyme A (hereinafter, coenzyme A may be abbreviated as "CoA") and 3-hydroxybutyrate CoA (hereinafter, sometimes abbreviated as "3-HB-CoA") using a genetically engineered microorganism. Specifically, the gene regions (icmA and icmB) encoding mutase, an enzyme that catalyzes the catabolism of 3-HB-CoA to 2-HIBA-CoA, are amplified by PCR from the genomic DNA of Aquincola tertiarycarbonis, and the two are ligated. Subsequently, the ligation product is amplified by PCR, and the resulting PCR fragment is introduced into a plasmid vector to obtain an expression vector. It has been reported that a genetically modified strain capable of producing 2-HIBA from a suitable carbon source can be obtained by genetically modifying Ralstonia eutropha with the expression vector. The parent strain, Ralstonia eutropha, is a microorganism that is originally capable of producing 3-HB-CoA from a suitable carbon source such as gluconic acid or fructose by the action of endogenous enzymes. Therefore, a genetically modified strain in which an exogenous mutase has been introduced into the parent strain can convert 3-HB-CoA to 2-HIBA-CoA, which is then hydrolyzed by the endogenous enzyme to produce 2-HIBA. In the examples described in Patent Document 1, it was confirmed that 0.72 mmol/kg of 2-HIBA was produced from a medium containing 15 g/L of sodium gluconate as a carbon source, and similar production of 2-HIBA was also confirmed when fructose was used as a carbon source.

Patent Document 2 reports that by combining icmA and icmB, which are gene regions encoding a mutase derived from Methylibium petroleiphilum, with phaA and phaB, which are gene regions encoding acetyl-CoA acetyltransferase and 3-hydroxybutyrate CoA dehydrogenase derived from Ralstonia eutropha, and introducing them into a plasmid vector to obtain an expression plasmid, and then genetically engineering the Escherichia coli MG1655 strain with the expression vector, a genetically modified strain capable of producing 2-HIBA from a suitable carbon source can be obtained, as in Patent Document 1. The 2-HIBA obtained in this manner can be converted to methacrylic acid by a dehydration reaction.

Special table number 2011-525367 Special table number 2012-500641

In order to establish the production of useful substances using genetic engineering techniques as an industrial process, it is necessary to culture microorganisms genetically engineered to produce the useful substances in large quantities at low cost. To do this, it is necessary to optimize the culture medium, culture conditions, culture equipment, etc. However, most microorganisms, including the genetically modified strains constructed in Patent Documents 1 and 2, prefer a low-salt environment with a NaCl concentration close to 0M as their preferred growth environment, and also prefer a neutral pH range as their preferred growth environment. Therefore, when culturing the genetically modified strains constructed in Patent Documents 1 and 2, it is necessary to sterilize or disinfect the culture medium and culture equipment directly used for culture, add antibiotics to the culture medium, etc., in order to prevent contamination by other microorganisms, which requires complicated operations and costs.

Therefore, the objective of the present invention is to provide a microorganism that can easily and inexpensively produce 2-HIBA from renewable resources, and a method for producing 2-HIBA.

As a result of intensive research conducted by the inventors to solve the above-mentioned problems, they have obtained a genetically modified strain capable of producing 2-HIBA easily and inexpensively from a suitable carbon source by introducing an acyl-CoA mutase gene (meaning "a nucleic acid molecule encoding a protein having acyl-CoA mutase activity") into Halomonas sp. KM-1 strain, a bacterium capable of growing in a halophilic, alkalophilic environment. Furthermore, they have found that by using this genetically modified strain, 2-HIBA can be produced easily and inexpensively. Based on these findings, the inventors have completed the present invention.

That is, the present invention relates to a microorganism capable of producing 2-hydroxyisobutyric acid, characterized in that it is a modified strain of Halomonas sp. KM-1 (deposit number FERM BP-10995) that expresses a protein having acyl-coenzyme A mutase activity derived from a thermophilic bacterium.

Another characteristic feature is that the acyl-coenzyme A mutase is methylmalonyl-coenzyme A mutase derived from Kyrpidia tusciae.

Another characteristic configuration is the Halomonas sp. KM-1/pOGE705KS-Ktmutase strain (accession number NITE P-03416).

According to the above characteristic configuration, the microorganism of the present invention is a parent strain of Halomonas sp. KM-1 that has been modified to express acyl-CoA mutase (meaning a "protein having acyl-CoA mutase activity") derived from a thermophilic bacterium. The microorganism of the present invention having the modification can produce 2-HIBA from an appropriate carbon source. Since such 2-HIBA can be easily chemically converted to methacrylic acid, etc., it is possible to supply methacrylic acid, etc. from renewable resources by using the microorganism of the present invention.

The microorganism of the present invention, like the parent strain KM-1, is halophilic and alkalophilic, and can therefore be suitably cultured even under conditions of relatively high salt concentration or alkaline conditions. Therefore, by culturing under suitable conditions, the risk of contamination and proliferation of other microorganisms can be largely eliminated. Therefore, culture can be performed in a non-sterile system, including culture using seawater, and there is no need to sterilize the medium or culture device during culture, making it possible to culture more easily and inexpensively than in the past.

The present invention also relates to a method for producing 2-hydroxyisobutyric acid, the method comprising a culture step of culturing the microorganism of the present invention in a medium containing a carbon source and inorganic salts, and a recovery step of recovering 2-hydroxyisobutyric acid from the culture obtained in the culture step.

According to the above-mentioned characteristic configuration, the method for producing 2-HIBA of the present invention uses the microorganism of the present invention having the above-mentioned excellent properties, so that 2-HIBA can be produced easily and inexpensively from renewable resources.

FIG. 1 is a flow diagram showing a 2-HIBA production pathway by a microorganism according to an embodiment of the present invention. Schematic diagram showing the structure of the methylmalonyl-CoA mutase operon derived from Kilpidia tussui, selected as an example of a gene introduced into the microorganism according to this embodiment. 1 is a plasmid map showing an outline of pBBR122ΔCm, which is a pBBR12 vector in which the drug resistance marker (CmR) has been removed. 1 is a plasmid map showing an outline of a plasmid vector (IPTG-inducible expression vector) introduced into the KM-1/pOGE705KS-Ktmutase strain, which is an example of a microorganism according to this embodiment. FIG. 2 is an electrophoretic diagram showing the results of confirming by PCR that the KM-1/pOGE705KS-Ktmutase strain, which is an example of a microorganism according to this embodiment, is a genetically modified strain into which an acyl-CoA mutase gene has been introduced.

The following describes an embodiment of the present invention in detail. However, the present invention is not limited to this embodiment.

[Microorganisms]
The microorganism according to the present embodiment is a modified strain having Halomonas sp. KM-1 strain (hereinafter, sometimes abbreviated as "KM-1 strain") as a parent strain. The microorganism has been modified to express acyl-CoA mutase derived from a thermophilic bacterium, which is not expressed in the KM-1 strain, and has the ability to produce 2-HIBA.

The parent strain, KM-1, was deposited on July 10, 2007 at the National Institute of Advanced Industrial Science and Technology (Chuo 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566) under the accession number FERM P-21316. This strain has now been transferred to an international depository, and its accession number is FERM BP-10995. The 16S rRNA gene of the KM-1 strain has been registered with the DDBJ under the accession number AB477015.

The microorganism according to this embodiment is preferably a genetically modified strain, into which an acyl-CoA mutase gene derived from a thermophilic bacterium has been introduced. Acyl-CoA mutase is an intramolecular transferase that catalyzes the catabolic reaction between 3HB-CoA and 2-HIBA-CoA. The acyl-CoA mutase is derived from a thermophilic bacterium, which is a bacterium with an optimum growth temperature of 45°C or higher, preferably 55°C or higher. Enzymes derived from thermophilic bacteria are highly heat-resistant and often resistant to alkalis, acids, denaturants, etc., and have been reported to have high industrial utility. An example of a thermophilic bacterium is Kirpidia tussui.

An example of an acyl-CoA mutase gene derived from a thermophilic bacterium is the methylmalonyl-CoA mutase gene derived from Kirpidia tussii DSM2912 strain (Applied and Environmental Microbiology, 2015 July, 81(14), 4564-4572). An example of the base sequence is shown in SEQ ID NO: 1 (methylmalonyl-CoA mutase operon: MeaB + large subunit + small subunit). The amino acid sequences encoded by the base sequence are shown in SEQ ID NOs: 2 to 4 (methylmalonyl-CoA mutase operon: SEQ ID NO: 2: MeaB, SEQ ID NO: 3: large subunit, SEQ ID NO: 4: small subunit). In addition, the acyl-CoA mutase gene may contain a base sequence that shows at least 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the base sequence shown in SEQ ID NO: 1, so long as it encodes a protein having acyl-CoA mutase activity. Furthermore, the acyl-CoA mutase gene may encode a protein having acyl-CoA mutase activity that has an amino acid sequence that shows at least 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 2 to 4.

An example of a microorganism according to this embodiment is the KM-1/pOGE705KS-Ktmutase strain shown in the following examples. This strain is a genetically modified strain in which the acyl-CoA mutase gene derived from the Kilpidia tussui DSM2912 strain shown in SEQ ID NO: 1 has been introduced into the parent strain KM-1, and has been deposited on February 17, 2021 at the National Institute of Advanced Industrial Science and Technology (Chuo 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566) under the accession number NITE P-03416.

(Physiological and biochemical characteristics)
The microorganism according to this embodiment has the same physiological and biochemical characteristics as the parent strain KM-1 strain. The microorganism can selectively use both oxidative and anaerobic metabolism, can grow regardless of the presence or absence of free oxygen, and tends to grow more easily in the presence of free oxygen, i.e., has the properties of a so-called facultative anaerobe.

The microorganism in this embodiment is a halophilic bacterium, and its optimal growth condition is a salt concentration of about 0.1 to 2.5 M, but it can also grow in the absence of salt.

The microorganisms according to this embodiment typically grow best in conditions of pH 5 to 12, and particularly pH 8 to 11, and can grow even in alkaline environments.

The microorganisms of this embodiment typically grow best at temperatures between 20 and 37°C, and particularly between 30 and 37°C.

The microorganisms according to this embodiment can be cultured suitably even under conditions of relatively high salt concentration or alkaline conditions. Therefore, by culturing under suitable conditions, the risk of contamination and proliferation of other microorganisms can be largely eliminated. Therefore, culture can be performed in a non-sterile system, including culture using seawater, and there is no need to sterilize the culture medium or culture device during culture, making it possible to culture more simply and inexpensively than in the past.

(2-HIBA production ability)
The microorganism according to the embodiment has the ability to produce 2-HIBA from a suitable carbon source. Hereinafter, the ability of the microorganism to produce 2-HIBA will be described based on FIG. 1. It has been reported that in the parent strain KM-1, a series of enzymes, namely, PhaA, PhaB, and PhaC, form a PHB synthesis pathway (see JP 2019-176838 A, etc.). Herein, PhaA is also called β-ketothiolase (acetyl CoA acetyltransferase) and converts acetyl CoA to acetoacetyl CoA. Acetyl CoA is produced, for example, through the aerobic metabolic process of pyruvic acid produced in the assimilation process of various carbon sources. PhaB is also called acetoacetyl CoA reductase (3-hydroxybutyrate CoA dehydrogenase) and reduces acetoacetyl CoA to (R)-3-hydroxybutyrate CoA (hereinafter, sometimes referred to as "(R)-3HB-CoA"). PhaC, also called polyhydroxyalkanoate polymerase, polymerizes (R)-3HB-CoA to synthesize polyhydroxybutyrate (hereinafter, sometimes referred to as "PHB"). PHB is further decomposed by PhaZ, also called polyhydroxyalkanoate depolymerase, into (R)-3-hydroxybutyrate (hereinafter, sometimes referred to as "(R)-3HB") with a degree of polymerization of 1 to 5.

The microorganism according to this embodiment is a parent strain of the KM-1 strain that has been modified to express acyl-CoA mutase, and as shown in FIG. 1, acyl-CoA mutase catalyzes the catabolic reaction between (R)-3HB-CoA and 2-HIBA-CoA. Therefore, unlike the KM-1 strain, this microorganism can produce 2-HIBA-CoA from various suitable carbon sources via (R)-3HB-CoA. Next, 2-HIBA-CoA is hydrolyzed to 2-HIBA and CoA-SH by endogenous thioesterase, and 2-HIBA is released from the body of the microorganism by an endogenous 2-HIBA exporter.

Since 2-HIBA can be easily converted chemically into methacrylic acid and other substances, the use of the microorganisms of the present invention makes it possible to supply methacrylic acid and other substances from renewable resources.

(Production method)
The microorganism according to this embodiment can be prepared by introducing an acyl-CoA mutase gene into the KM-1 strain and expressing the gene. The introduction of the acyl-CoA mutase gene into the KM-1 strain can be carried out using a known genetic engineering technique. For example, the acyl-CoA mutase gene is inserted into an appropriate vector to prepare an expression vector, and the expression vector is introduced into the KM-1 strain. There is no particular limitation on the vector that can incorporate a foreign nucleic acid molecule and can autonomously replicate in the KM-1 strain. The expression vector incorporates the foreign nucleic acid molecule so that it can express its function, and contains other known base sequences necessary for functional expression. For example, the expression vector usually contains a promoter sequence, a replication origin, a restriction enzyme cleavage site, a transcription terminator sequence, and the like, and can further contain a leader sequence, a signal sequence, a ribosome binding sequence, and a marker sequence.

Suitable vectors include plasmid vectors (pUC series, pBluescript series, pBR series, pGEX series, etc.), phage vectors (lambda gt10, lambda gt11, lambda ZAP, etc.), cosmid vectors, and viral vectors (vaccinia virus, baculovirus, etc.), but are not limited to these, and any known vector can be used. Plasmid vectors are preferred.

Suitable promoter sequences may be either inducible or constitutive promoters, but are preferably inducible promoters. An inducible promoter is one that can induce expression of a target gene in the presence or absence of a specific factor, while a constitutive promoter is one that can constitutively express a target gene. Specific suitable promoters include lac promoter, tac promoter, trp promoter, T7 promoter, etc. However, the present invention is not limited to these, and any known promoter sequence can be used.

In general, when a foreign nucleic acid molecule is introduced, the foreign nucleic acid molecule may adversely affect the growth of the microorganism. Therefore, in this embodiment as well, it is preferable to suppress the expression of the thermophilic acyl-CoA mutase gene until the KM-1 strain genetically modified with the thermophilic acyl-CoA mutase gene grows sufficiently, and then express the gene when the strain has grown to a certain extent. For example, the expression of the thermophilic mutase gene can be controlled by using the lactose operon, and the expression of the gene can be induced by isopropyl β-D-thiogalactopyranoside (hereinafter sometimes abbreviated as "IPTG").

The marker sequence confers phenotypic selection in the microorganism into which the expression vector has been introduced, and examples thereof include nucleic acid sequences that code for drug resistance genes, fluorescent proteins, and genes for enzymes that catalyze color reactions. Specific examples include the spectinomycin resistance gene, the kanamycin resistance gene, the chloramphenicol resistance gene, and the ampicillin resistance gene.

The amino acid sequence information and base sequence information of the acyl-CoA mutase gene derived from a thermophilic bacterium are disclosed in databases such as Kyoto Encyclopedia of Genes and Genomes (KEGG), GenBank, DDBJ, and EMBL-Bank, and therefore the gene can be prepared based on the sequence information by known genetic engineering techniques or chemical synthesis techniques. As a genetic engineering technique, for example, a suitable probe can be prepared based on the sequence information, and the desired acyl-CoA mutase gene derived from a thermophilic bacterium can be obtained by hybridization, preferably from chromosomal DNA derived from an organism such as a thermophilic bacterium, and cDNA synthesized from mRNA by reverse transcription reaction. The gene can also be obtained by PCR using a primer prepared based on the sequence information. The probe and primer used here can be an oligonucleotide consisting of 10 or more, preferably 15 or more, more preferably about 20 to 50 bases, containing a sequence complementary to the acyl-CoA mutase gene derived from the desired thermophilic bacterium. For example, the primers can be designed using primer design support software or the like so as to amplify the desired region. Here, complementary means that the probe or primer and the base sequence of the target gene can specifically bind to each other according to the base pairing rules to form a stable double-stranded structure. Here, not only complete complementarity but also partial complementarity in which only some bases match according to the base pairing rules is acceptable as long as the probe or primer and the base sequence of the target gene are sufficient to form a stable double-stranded structure with each other. The probe and primers can be prepared based on a conventional method. For example, a chemical synthesis method or, if the target gene has already been obtained, a restriction enzyme fragment thereof can be used. As a chemical synthesis method, a known DNA synthesis method such as the phosphoramidite method can be used.

The acyl-CoA mutase gene can be inserted into a vector by, for example, cleaving the desired acyl-CoA mutase gene with an appropriate restriction enzyme, and inserting and linking it into a restriction enzyme cleavage site or a multicloning site of an appropriate vector, but is not limited to this and any known method can be used.

The constructed expression vector can be introduced into the KM-1 strain by any known method, including, but not limited to, the calcium chloride method, electroporation, liposome infection, and microinjection.

Selection of the desired genetically modified strain can be performed based on known methods. For example, it can be performed based on the presence or absence of expression of a marker sequence included in an expression vector. When a drug resistance gene is used as the marker sequence, it can be performed by culturing in a medium containing a drug corresponding to the drug resistance gene. In addition, introduction of the acyl-CoA mutase gene can be confirmed by PCR using primers containing a base sequence characteristic of the acyl-CoA mutase gene.

[Method of producing 2-HIBA]
The method for producing 2-HIBA according to this embodiment is as follows:
(1) a culturing step of culturing a microorganism according to this embodiment in a medium containing a carbon source and an inorganic salt;
(2) a recovery step of recovering 2-HIBA from the culture solution obtained in the culture step.

(Step (1): Culture step)
Step (1) in the method for producing 2-HIBA according to this embodiment is a step of culturing the microorganism according to this embodiment in a medium containing a carbon source and inorganic salts.

<Microorganisms>
The microorganism used in step (1) is the microorganism according to the present embodiment. The microorganism according to the present embodiment can be cultured in the same manner as the parent strain KM-1. It is cultured in a medium containing a carbon source and inorganic salts. The "carbon source" and "carbon source" will be described later in the section "Medium".

<Culture medium>
The medium used in the above step (1) contains inorganic salts and a carbon source, and is preferably a liquid medium. The pH of the medium is not particularly limited, but is preferably a pH that satisfies the growth conditions of the microorganism of this embodiment, specifically, within a range of about pH 5 to 12. More preferably, it is within a range of about pH 8 to 11. By using an alkaline medium, contamination with other microorganisms and the like can be effectively prevented without the need for special measures.

The inorganic salts contained in the medium used in step (1) are not particularly limited. For example, phosphates, nitrates, carbonates, sulfates, and metal salts such as sodium, magnesium, potassium, manganese, iron, zinc, copper, and cobalt can be used.

_For example, when a sodium salt is used as _the inorganic salt, NaCl, _NaNO3 , _NaNO2 , NaHCO3, _Na2CO3 , _Na2HPO4 , _{NaH2PO4 ,} etc. can be used. When _a potassium salt is used as the inorganic salt, _KNO3 , _KNO2 , _K2HPO4 , _{KH2PO4 , etc.} can be used.

It is preferable to use these inorganic salts as compounds that serve as nitrogen and phosphorus sources for the strain of this embodiment.

The nitrogen source may be, but is not limited to, nitrates, nitrites, ammonium salts, urea, glutamic acid, etc. For example, nitrogen-containing compounds such as NaNO ₃ , NaNO ₂ , and NH ₄ Cl may be used.

The amount of nitrogen source used can be appropriately set within a range that achieves the purpose of producing 2-HIBA without affecting the growth of the microorganism according to this embodiment. Specifically, the amount of nitrate used should usually be at least about 250 mg per 100 mL of culture medium at the beginning of the culture, more preferably at least about 1000 mg, and even more preferably at least about 1250 mg.

The phosphorus source may be, but is not limited to, phosphate, monohydrogen _phosphate , dihydrogen phosphate _, etc., and examples of compounds _that can be used include _Na3PO4 , _Na2HPO4 , _{NaH2PO4 , K3PO4} , _{K2HPO4 , and KH2PO4} .

The amount of phosphorus source used may be set appropriately from the same viewpoint as the amount of nitrogen source used above. Specifically, the amount of dihydrogen phosphate is usually within the range of about 50 to 400 mg per 100 mL of medium, and preferably about 100 to 200 mg.

These inorganic salts may be used alone or in combination of two or more.

The inorganic salts are usually used at a total concentration within the range of about 0.1 to 2.5 M, preferably about 0.2 to 1.0 M, and more preferably about 0.2 to 0.5 M. By using a medium with a relatively high salt concentration, contamination by other microorganisms can be effectively prevented without the need for special measures.

The carbon source contained in the medium used in step (1) is not particularly limited, but may be Examples of the sugars include disaccharides such as sucrose, lactose, maltose, trehalose, turanose, and cellobiose; pentoses such as ribulose, xylulose, ribose, arabinose, xylose, lyxose, and deoxyribose; hexoses such as psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose, gulose, idose, galactose, and talose; sugar alcohols such as erythritol, glycerin, mannitol, sorbitol, and xylitol; tryptone, yeast extract, soluble starch, ethanol, n-propanol, acetic acid, and acetates such as sodium acetate; propionic acid, waste glycerol, waste molasses, wood saccharification solution, pyruvic acid, acetyl CoA, citric acid, α-ketoglutaric acid, succinic acid, fumaric acid, malic acid, oxaloacetic acid, and glutamic acid, but are not limited thereto.

These carbon sources can be used alone or in combination of two or more. The amount of carbon source used is not particularly limited, and it is usually sufficient to use the carbon source at a concentration within the range of about 0.0056 to 0.1666, preferably about 0.0278 to 0.1389 M, and more preferably about 0.056 to 0.111 M.

Furthermore, the medium can contain a factor corresponding to the gene marker contained in the expression vector so that the microorganism according to this embodiment can be selectively cultured. For example, when a drug resistance gene is used as the marker sequence, a drug corresponding to the drug resistance gene can be contained.

As the medium used in step (1), preferably, the SOT medium used in the examples can be used.

<Culture method>
The culture method in step (1) is not particularly limited. One embodiment of the culture method is described below.

The culturing step in step (1) is not particularly limited as long as the conditions are such that the microorganism according to this embodiment can grow and produce 2-HIBA from an appropriate carbon source. Preferably, the culturing step can be performed by carrying out an aerobic fermentation step followed by a microaerobic fermentation step.

Specifically, the aerobic fermentation process involves inoculating the microorganism of this embodiment into approximately 5 mL of medium, and pre-culturing the microorganism while shaking overnight, usually at approximately 30 to 37°C and at an agitation speed of approximately 120 to 250 rpm. The bacterial cells obtained by pre-cultivation are then diluted approximately 100-fold in a medium contained in an Erlenmeyer flask, fermenter, jar fermenter, or the like, and main culture is carried out.

Main culture can usually be performed at about 20 to 45°C, but it is preferable to perform the culture at about 30 to 42°C. The culture environment should be one in which the medium is exposed to air, and a method of actively blowing oxygen-containing gas onto the surface of the medium or blowing gas into the medium may be used.

In the aerobic fermentation process, the microorganism of this embodiment may be aerobically cultured under such culture conditions. Specifically, the dissolved oxygen concentration in the medium during aerobic culture is not particularly limited, but is usually 0 mg/L or more, and preferably 7 mg/L or more.

The culture method in the aerobic fermentation process includes, but is not limited to, batch culture, semi-batch culture, continuous culture, etc., but in order to efficiently produce 2-HIBA, long-term continuous culture is also possible, taking into account that the possibility of contamination with other bacteria during the culture of the microorganism according to this embodiment is extremely low. The culture environment is also not particularly limited, and may be a non-sterile environment or a sterile environment.

In the microaerobic fermentation process, the microorganism of this embodiment may be cultured in a culture medium adjusted and maintained in an alkaline region, for example, at pH 8.0 or higher, under conditions where oxygen is not actively supplied. If the culture is continued without actively supplying oxygen to the microorganism of this embodiment, the oxygen in the system is consumed and the system becomes close to anoxic, but since an environment with an oxygen concentration of several percent, which is not absolutely anaerobic, is maintained, an environment suitable for microaerobic culture can be maintained.

If the culture is continued, the pH of the medium tends to decrease due to the production of organic acids. The pH of such a medium can be checked appropriately using a known pH measuring device or a jar fermenter with an attached pH measuring device.

The term "adjust and maintain" means that the pH is maintained at a suitable level by adding a pH adjuster while checking the pH as described above.

The pH adjusted and maintained in the microaerobic process is preferably 8.0 or higher. This allows the productivity of 2-HIBA by the microorganism of this embodiment to be maintained at a high level.

Examples of pH adjusters include, but are not limited to, hydroxides, carbonates, and bicarbonates. More specifically, examples include sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium carbonate, potassium carbonate, calcium carbonate, magnesium carbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, and aqueous ammonia.

(Step (2): Recovery step)
Step (2) in the method for producing 2-HIBA according to this embodiment is a step of recovering 2-HIBA from the culture obtained in the above step (1). The culture means the medium after the culture in step (1) and the microorganisms grown by the culture, and may be either one of them.

2-HIBA may also be recovered as a salt that reacts with ions derived from inorganic salts contained in the culture.

Ions derived from inorganic salts are not particularly limited, but examples include alkali metal ions such as sodium ions and potassium ions, alkaline earth metal ions such as calcium ions, and cations such as magnesium ions, cobalt ions, zinc ions, iron ions, copper ions, molybdenum ions, ammonium ions, and manganese ions.

The recovery in step (2) can be carried out after the culture step in step (1) is stopped. The means for stopping the culture is not particularly limited, and examples include a method of sterilizing the microorganism to be cultured according to this embodiment by heating or acid treatment, or a method of separating the microorganism from the medium using a known solid-liquid separation means. The timing for stopping the culture step can be appropriately determined by confirming the presence of 2-HIBA in the culture. The means for confirming the presence of 2-HIBA in the culture can be changed depending on the type of microorganism used, the medium components, the culture conditions, etc., and can be appropriately determined taking these factors into consideration. For example, the culture can be sampled over time and subjected to analytical means such as LC-MS or HPLC to determine the time to stop the culture.

When 2-HIBA is present in the medium of the culture obtained in step (1), the recovery step in step (2) can be carried out by, for example, using a known separation means to recover the medium containing 2-HIBA as a liquid fraction. The specific separation means is not particularly limited, and solid-liquid separation means such as centrifugation and filtration can be used alone or in appropriate combination. Furthermore, when 2-HIBA is present in the microorganisms of the culture obtained in step (1), the recovery step can be carried out by disrupting the microorganisms using a known disruption means, and then recovering the liquid fraction containing 2-HIBA using a known separation means. The specific disruption means is not particularly limited, and enzymatic disruption means such as lysozyme treatment, or physical disruption means such as ultrasonic treatment, freeze-thawing, and osmotic shock can be used alone or in appropriate combination.

The recovered liquid fraction may be purified by known purification means. There are no particular limitations on the specific purification means, and known purification means such as desalting, dialysis, gel filtration, and various types of chromatography such as hydrophobic, anionic, cationic, and affinity chromatography may be used alone or in appropriate combination. It is also possible to add a lower alcohol to the recovered liquid fraction, subject it to an esterification reaction, and purify it as a 2-HIBA ester by distillation or the like.

By using the method for producing 2-HIBA according to this embodiment, the produced 2-HIBA can be converted to methacrylic acid by a dehydration reaction. Furthermore, it can be converted to a methacrylic acid ester by an esterification reaction, and various methacrylic acid derivatives can be produced. Furthermore, polymethacrylic acid and polymethacrylic acid derivatives can be produced by radical polymerization of methacrylic acid and methacrylic acid esters.

The present invention will be described in more detail below with reference to examples. Note that the present invention is not limited to the following examples.

＜Overview of Experimental Methods＞
a. PCR in gene cloning
PCR for gene cloning was performed using PrimeSTAR HS DNA Polymerase in a thermal cycler (T100 Thermal Cycler: BIO-RAD) to amplify the target gene fragment. After heating at 98°C for 2 minutes, the temperature profile was 30 cycles of 98°C for 15 seconds, 55°C for 10 seconds, and 72°C for 1 minute/kb.

b. PCR for confirming genetically modified strains
PCR for confirming the genetically modified strain was performed by amplifying the target gene fragment using Quick Taq HS DyeMix with a thermal cycler (T100 Thermal Cycler: BIO-RAD). After heating at 95°C for 3 minutes, the temperature profile was 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute/kb.

c. Purification of PCR products The PCR amplified products were purified using a QIAquick Gel Extraction Kit according to the manufacturer's manual to purify DNA fragments from 50 μL of the PCR reaction product. The DNA was eluted with 50 μL of purified water.

Electrophoresis was performed using a 1% agarose gel. After staining DNA with ethidium bromide, the electrophoretic image was photographed using Gel Viewer (WAKO).

e. DNA extraction from electrophoresis gel The DNA fragment was purified from the agarose gel using QIAquick Gel Extraction Kit according to the manufacturer's manual. DNA elution was performed with 20 μL of purified water.

f. Ligation reaction
The DNA fragments were ligated using Ligation high according to the manufacturer's manual.

<Preparation of a modified strain capable of producing 2-HIBA>
1. Selection of acyl-CoA mutase gene As an acyl-CoA mutase capable of catabolizing 3-HB-CoA to 2HIBA-CoA that can be produced by the KM-1 strain, methylmalonyl-CoA mutase derived from Kilpidia tussui was selected (Applied and Environmental Microbiology, 2015 July, 81(14), 4564-4572). Sequence information of the selected Kilpidia tussui methylmalonyl-CoA mutase gene operon was obtained from the KEGG database. The genome structure of K. tussui forms an operon consisting of MeaB (SEQ ID NO: 2), which is an HCM (2-hydroxyisobutylyl CoA mutase)-associated G protein chaperone, and the large and small subunits of methylmalonyl CoA mutase (SEQ ID NO: 3 and SEQ ID NO: 4) (see FIG. 2). The number of amino acid residues encoded by each gene in K. tussui is 312 for MeaB, 563 for the large subunit, and 132 for the small subunit.

2. Preparation of acyl-CoA mutase expression vector An IPTG-inducible acyl-CoA mutase expression vector (pOGE705KS/PlacZ-Kt mutase) based on pBBR122 was prepared as follows.

In order to clone the acyl-CoA mutase gene operon selected above, the genomic DNA of Kilpidia tussui was purified. First, Kilpidia tussui was cultured in 3 mL of 294 medium at 50°C for 3 days. The cells were collected by centrifugation, and the genomic DNA was extracted using Quick Gene DNA tissue kit S. A gene fragment (Kt mutase) from the initiation codon of the MeaB gene in Kirpidia tussui to the termination codon of the small subunit of acyl-CoA mutase was amplified by PCR using a primer pair (Kt 468-470NdeI F1: CAGTAGACATATGCAAGAGCTTCTCTCGCGAT (SEQ ID NO: 5) and Kt 468-470SacI R1: AGTCGAGCTCTCAATCCCGATCCGAAACC (SEQ ID NO: 6)). This Kt mutase was cloned into the NdeI/SacI site of pHSG298 (Takara) to construct pHSG298-Kt mutase.

Sequencing revealed that the cloned Kt mutase sequence was exactly identical to the reported sequence. Next, in order to place a lacIQ-PlacZ fragment consisting of lacIQ and lacZ promoters enabling IPTG induction upstream of the Kt mutase of pHSG298-Kt mutase, the lacIQ-PlacZ fragment was amplified by PCR using the E. coli (DH5α) genome as a template and a primer pair (PlacIQ-XbaI: AGTCTCTAGACCATCGAATTAAGCAAAACC (SEQ ID NO: 7) and PlacIQ-NdeI: CAGTAGACATATGTGTTTCCTGTGAAATTGTT (SEQ ID NO: 8)) and inserted into the XbaI/NdeI site of pHSG298-Kt mutase to obtain pHSG298-lacIQ-PlacZ-Kt. A mutase was constructed. Furthermore, in order to replace the chloramphenicol resistance gene of pHSG298-lacIQ-PlacZ-Kt mutase with a spectinomycin resistance gene, a SpeR fragment containing a spectinomycin resistance gene was amplified by PCR using a primer pair (SpecSacIIFBglII: CAGTAGATCTCCGCGGACCAGCCAGGACAG (SEQ ID NO: 9) and SpecSacIIRBglII: CAGTAGATCTCCGCGGTTTTCTACGGGGTC (SEQ ID NO: 10)) with the pCR8/GW/TOPO vector as a template, and introduced into the SacII site of pHSG298-lacIQ-PlacZ-Kt mutase to obtain pHSG298-lacIQ-PlacZ-Kt. Mutase/SpecR was constructed.

The lacIQ-PlacZ-Kt mutase/SpecR fragment was excised from the obtained pHSG298-lacIQ-PlacZ-Kt mutase/SpecR by digestion with BglII and inserted into the BglII site of pBBR122ΔCm (see Figure 3), which is based on the pBBR122 vector that can autonomously replicate in a wide range of Gram-negative bacteria, to create an IPTG-inducible acyl-CoA mutase expression vector (pOGE705KS/PlacZ-Kt mutase) (see Figure 4).

Here, pBBR122ΔCm was constructed by using pBBR122 as a template and a primer pair (pBBR122-KmRBglIIF: AGTCAGATCTCCTCACGGCGGCGAGTGCGG (SEQ ID NO: 11) and pBBR122-KmRBglIIR: CAGTAGATCTCTCGGGGGGCAGGCGGGCGG (SEQ ID NO: 12)) to amplify a DNA fragment by PCR, treat it with BglII, and then self-ligate it.

As shown in Figure 4, the introduced acyl-CoA mutase gene is in a state where the lactose repressor (lacIq) binds to the lactose operator (lacO) and expression from the lacZ promoter is suppressed. When IPTG is added, the lactose repressor binds to IPTG and changes its three-dimensional structure, dissociating from the lactose operator and inducing expression of the mutase gene from the lacZ promoter. The expression vector also uses a spectinomycin resistance gene, a drug resistance gene, as a marker sequence.

3. Introduction of acyl-CoA mutase expression vector pOGE705KS-Ktmutase was introduced into KM-1 strain competent cells by electroporation to obtain the genetically modified strain KM-1/pOGE705KS-Ktmutase strain. Electroporation was performed using Gene Pulser Xcell (BIO-RAD) under the following conditions: voltage: 2.5 kV, resistance: 200 Ω, capacitance: 25 μF, cell width: 2 mm. Transformation of E. coli was performed using DH5α Jet competent cells and JM109 competent cells according to the manufacturer's manual.

4. Confirmation of genetically modified strain It was confirmed by PCR that the KM-1/pOGE705KS-Ktmutase strain was a genetically modified strain into which the acyl-CoA mutase gene had been introduced. In detail, 1 mL of SOT medium was added to the bacterial cell liquid after electroporation, and the cells were cultured with shaking at 30°C for 1 hour. The cultured bacterial cells were collected by centrifugation and spread on a SOT plate containing 50 μg/mL or 100 μg/mL spectinomycin. After static culture at 30°C for 2 to 3 days, it was confirmed by PCR that the 6 colonies formed contained the acyl-CoA mutase gene. For PCR, a primer pair for amplifying the entire sequence of the introduced acyl-CoA mutase gene (Kt Ptac468-470XbaI F1: AGTCTCTAGAGAGCTGTTGACAATTAATC (SEQ ID NO: 13), Kt Ptac468-470XbaI R1: CAGTTCTAGAAACGCAAAAAGGCCATCCG (SEQ ID NO: 14)) and a primer pair for amplifying the bdh2 internal sequence of the parent strain KM-1 (bdh2 F1: ACTTCAACGATCAGGCCAG (SEQ ID NO: 15), bdh2 R1: ATGCCGTGCTTGGCACTGA (SEQ ID NO: 16)) were prepared. PCR was performed using the primer pair. The amplified fragment was then subjected to electrophoresis. The results are shown in Figure 5. As a result, it was confirmed that the acyl-CoA mutase gene (shown as Kt.mutase in the figure) was introduced into the KM-1/pOGE705KS-Ktmutase strain.

4. Production of 2-HIBA The KM-1/pOGE705KS-Ktmutase strain was cultured in the presence of IPTG to induce expression of the acyl-CoA mutase gene, and the productivity of 2-HIBA was evaluated.

In detail, 30 mL of SOT medium was placed in a baffled 200 mL flask, and the KM-1 strain and the KM-1/pOGE705KS-Ktmutase strain were inoculated to an OD of 0.1. The culture conditions were a culture temperature of 33°C and a shaking speed of 200 rpm. Expression induction with IPTG was performed by adding IPTG to a final concentration of 1 mM when OD reached 0.6.

<2-HIBA production test by flask culture>
Plasco shaking culture was performed using SOT medium (see JP 2013-081403 A) containing sucrose as a carbon source. The composition of the SOT medium used in this example is shown in Table 1. As a result, while no production of 2-HIBA was confirmed in the KM-1 strain, production of 16.5 mg/L of 2-HIBA was confirmed 48 hours after the start of culture in the KM-1/pOGE705KS-Ktmutase strain.

These results confirmed that the KM-1/pOGE705KS-Ktmutase strain has the ability to produce 2-HIBA. This can be understood as meaning that (R)-3HB-CoA is produced from an appropriate carbon source, which is then converted to 2-HIBA-CoA by the action of the introduced acyl-CoA mutase, which is then hydrolyzed to 2-HIBA and CoA-SH by endogenous thioesterase, and 2-HIBA is then released into the medium by the endogenous 2-HIBA exporter.

2-HIBA can be produced simply and inexpensively from renewable resources, and 2-HIBA can be easily converted chemically into methacrylic acid. This technology is expected to be applicable to any industrial application that utilizes 2-HIBA and methacrylic acid, etc.

Claims (3)

A microorganism capable of producing 2-hydroxyisobutyric acid,
A modified strain of Halomonas sp. KM-1 (deposit number FERM BP-10995) expressing acyl-coenzyme A mutase derived from a thermophilic bacterium ,
The acyl-coenzyme A mutase is methylmalonyl-coenzyme A mutase derived from Kyrpidia tusciae . The microorganism according to claim 1, which is Halomonas sp. KM-1/pOGE705KS-Ktmutase strain (accession number NITE P-03416). A culturing step of culturing the microorganism according to claim 1 or 2 in a medium containing a carbon source and inorganic salts;
and a recovery step of recovering 2-hydroxyisobutyric acid from the culture obtained in the culture step.

Images