WO2023120538A1

WO2023120538A1 - Method for producing 4-hydroxybenzoic acid

Info

Publication number: WO2023120538A1
Application number: PCT/JP2022/046954
Authority: WO
Inventors: 篤生鶴谷; 宏征栗原; 和典園木; 雄大樋口
Original assignee: 東レ株式会社; 国立大学法人弘前大学
Priority date: 2021-12-21
Filing date: 2022-12-20
Publication date: 2023-06-29
Also published as: JPWO2023120538A1

Abstract

The present invention addresses the problem of providing a method for producing 4-hydroxybenzoic acid (4HBA) with high efficiency from a starting material such as a biomass, and provides a method for producing 4-hydroxybenzoic acid which includes the following steps (1) and (2). Step (1): a step for preparing an aqueous solution containing coumaric acid and ferulic acid; and step (2): a step for allowing a bacterium in which the expression of PobA enzyme is defective to act on the aqueous solution prepared in step (1) to metabolize the ferulic acid as a growth carbon source by the bacterium and convert the coumaric acid to 4-hydroxybenzoic acid by the bacterium.

Description

Method for producing 4-hydroxybenzoic acid

The present invention relates to a method for producing 4-hydroxybenzoic acid using biomass or the like as a raw material.

　4-Hydroxybenzoic acid (4HBA) is used not only as a monomer for liquid crystal polymers, but also as an antioxidant and as a raw material for agricultural chemicals. 4HBA is produced by the Kolbe-Schmidt reaction, in which carbon dioxide is reacted with petroleum-derived phenol alkali salt at high temperature and high pressure. It has become.

On the other hand, a method of producing hydroxycinnamic acids such as ferulic acid and coumaric acid from biomass is known, and it is disclosed that bagasse, which is sugarcane residue, can be used as a raw material (Patent Document 1). Moreover, a method of producing high-purity ferulic acid from a biomass extract and converting the obtained ferulic acid into vanillin is disclosed (Patent Document 2). Furthermore, it has been disclosed that 4HBA can be accumulated from coumaric acid by knocking out the pobA gene of Pseudomonas bacteria (Non-Patent Document 1). In this way, the possibility of producing 4HBA using biomass as a raw material has been suggested, but there have been no reports of a single, easy production of 4HBA from a biomass raw material, and commercial production has not been realized. Therefore, further technical development is required.

WO2017/170549 JP 2019-89778 A

The purpose of the present invention is to provide a method for highly efficient production of 4-hydroxybenzoic acid (4HBA) from raw materials such as biomass.

The inventor of the present invention focused on hydroxycinnamic acids contained in extracts from raw materials such as biomass and conducted extensive studies. The inventors also found that coumaric acid contained in the extract is converted to 4-hydroxybenzoic acid (4HBA) while metabolizing and growing as , and completed the present invention.

That is, the present invention includes the following [1] to [6].
[1] A method for producing 4-hydroxybenzoic acid, comprising the following steps (1) and (2).
Step (1): Preparing an aqueous solution containing coumaric acid and ferulic acid Step (2): Bacteria deficient in PobA enzyme expression are allowed to act on the aqueous solution prepared in Step (1), and the bacteria produce ferulic acid. as a growth carbon source, and converting the coumaric acid to 4-hydroxybenzoic acid by the bacteria [2] The step (1) includes alkali-treating the lignin-containing biomass to contain coumaric acid and ferulic acid. The method for producing 4-hydroxybenzoic acid according to [1], which is a step of obtaining an extract as the aqueous solution.
[3] The method for producing 4-hydroxybenzoic acid according to [1] or [2], wherein the aqueous solution prepared in step (1) has a sugar concentration of less than 5 g/L.
[4] Any one of [1] to [3], wherein the aqueous solution prepared in step (1) contains one or more organic acids selected from the group consisting of acetic acid, formic acid and lactic acid. A method for producing 4-hydroxybenzoic acid according to .
[5] The method for producing 4-hydroxybenzoic acid according to any one of [1] to [4], wherein the bacterium used in step (2) is Pseudomonas bacterium.
[6] The method for producing 4-hydroxybenzoic acid according to any one of [1] to [5], wherein in the step (2), the bacteria are allowed to act on the aqueous solution under conditions of pH 5-7.

In the present invention, ferulic acid contained in an aqueous solution (for example, an extract from raw materials such as biomass) is used as a carbon source for growth by bacteria lacking PobA enzyme expression (hereinafter sometimes referred to as "bacteria of the present invention"). As it is metabolized, coumaric acid in the same aqueous solution is converted to 4-hydroxybenzoic acid. That is, ferulic acid is an impurity in the production of 4-hydroxybenzoic acid and is removed by the bacterium of the present invention. In particular, alkaline extracts of lignin-containing biomass may also contain other impurities, such as organic acids and amino acids, which are also removed from the bacteria of the invention. Due to these effects, highly pure 4-hydroxybenzoic acid can be obtained with a small number of purification steps.

Although the present invention will be described in detail below, the present invention is not limited to these.

<Step (1)>
Step (1) is a step of preparing an aqueous solution containing coumaric acid and ferulic acid. The step (1) will be described below.

In the present invention, the aqueous solution containing coumaric acid and ferulic acid refers to an aqueous solution containing at least coumaric acid and ferulic acid. , an extract obtained by extracting coumaric acid and ferulic acid contained in a solid raw material. The extraction method from solid raw materials containing coumaric acid and ferulic acid is not limited as long as coumaric acid and ferulic acid can be efficiently extracted. Extraction with an alkaline aqueous solution is preferable because the properties of the extract are enhanced. In addition, since ferulic acid and coumaric acid are sometimes covalently bound to polysaccharides in the solid raw materials, in order to increase the extraction efficiency, a hydrolysis reaction is performed in a high-temperature alkaline aqueous solution for several hours to obtain ferulic acid. and coumaric acid are preferably eluted in an alkaline aqueous solution.

The solid raw material for obtaining the extract is preferably lignin-containing biomass. Lignin-containing biomass refers to biological resources that contain at least lignin components. Specific examples of lignin-containing biomass include herbaceous biomass such as bagasse, corn cob, switchgrass, napier grass, erianthus, corn stover, rice straw, and straw; woody biomass such as trees and waste building materials; biomass derived from aquatic environments such as algae and seaweed; grain husk biomass such as corn husks, wheat husks, soybean husks and rice husks.

In a preferred embodiment, step (1) is a step of treating lignin-containing biomass with alkali to obtain an extract containing coumaric acid and ferulic acid as the aqueous solution.

In the present invention, the alkali treatment is a treatment in which 0.1 to 1 g of an alkali component is added per 1 g weight of lignin-containing biomass and left at room temperature or under heating for a certain period of time. Examples of alkalis that can be used include sodium hydroxide, calcium hydroxide, and ammonia. The temperature of the alkali treatment may be set in the range of room temperature to 300° C. so that the amount of coumaric acid and ferulic acid extracted is maximized. The temperature of the alkali treatment is preferably 50°C or higher and lower than 100°C, more preferably 65°C or higher and 90°C or lower. The solid content concentration of the lignin-containing biomass in the alkali treatment is preferably adjusted to 1 to 25% by weight, more preferably 5 to 20% by weight, by adding water. Since coumaric acid and ferulic acid are dissolved in the alkaline extract after the alkali treatment reaction, the extract can be obtained by separating and removing the solids. Moreover, it is preferable to add an acid to neutralize or acidify the solid content before separating and removing the solid content. By neutralizing or acidifying with an acid, only the alkali-soluble lignin dissolved in the alkali extract can be selectively precipitated, and by separating and removing these as solids, the conversion efficiency of microorganisms can be improved. It has a boosting effect. The pH range after neutralization or acidification is preferably pH 3-7.5, more preferably pH 4.5-5.5. Within these pH ranges, coumaric acid and ferulic acid remain dissolved, and only alkali-soluble lignin can be selectively deposited and separated and removed. The method of solid-liquid separation is not particularly limited, but centrifugation using a screw decanter, etc.; membrane separation using a filter press, etc.; separation using a belt press, belt filter, natural sedimentation, etc.; Solid-liquid separation can be performed. The extract containing coumaric acid and ferulic acid obtained by the above operation may be adjusted in component concentration before being used in step (2).

In a preferred embodiment, step (1) is a step of subjecting bagasse to alkali treatment, followed by neutralization to remove solids to obtain an extract containing coumaric acid and ferulic acid as the aqueous solution.

The concentration of coumaric acid in the aqueous solution prepared in step (1) is, for example, 1-1000 mg/L, preferably 10-200 mg/L. The concentration of ferulic acid in the aqueous solution in step (1) is, for example, 1-1000 mg/L, preferably 10-200 mg/L, more preferably 10-50 mg/L. Since coumaric acid can be converted to 4-hydroxybenzoic acid (4HBA), the higher the concentration of coumaric acid, the better, but ferulic acid is a carbon source for growth and becomes an impurity if left unconsumed by microorganisms. Therefore, the ferulic acid concentration is preferably relatively lower than the coumaric acid concentration.

The aqueous solution prepared in step (1) may contain other aromatic compounds such as vanillic acid and vanillin in addition to coumaric acid and ferulic acid. These aromatic compounds are used in step (2) as a growth carbon source for the bacteria of the present invention. The vanillic acid concentration of the aqueous solution prepared in step (1) is, for example, 0-100 mg/L, preferably 0-10 mg/L. The vanillin concentration of the aqueous solution prepared in step (1) is, for example, 0-100 mg/m, preferably 0-10 mg/L. In addition, when the vanillin and/or vanillic acid concentration exceeds 100 mg/L, bacterial growth may be inhibited.

The aqueous solution prepared in step (1) usually does not contain 4-hydroxybenzoic acid (4HBA).

The aqueous solution prepared in step (1) may contain sugar, and the sugar concentration in that case is preferably less than 5 g/L, more preferably 1 g/L or less. The lower limit of the sugar concentration of the aqueous solution prepared in step (1) is zero. When the sugar concentration of the aqueous solution prepared in step (1) is 5 g/L or more, the 4HBA production rate and the 4HBA yield from coumaric acid decrease. This is because the bacterium of the present invention preferentially consumes sugar as a carbon source. Here, sugar means monosaccharide (eg, glucose, fructose, xylose, mannose, arabinose, etc.), disaccharide (eg, sucrose, etc.), oligosaccharide, polysaccharide, and the like. The sugar is preferably one or two or more sugars selected from monosaccharides and disaccharides. When the aqueous solution prepared in step (1) contains two or more sugars, the sugar concentration means the total concentration of the two or more sugars.

The aqueous solution prepared in step (1) preferably contains one or more organic acids selected from the group consisting of acetic acid, formic acid and lactic acid. These organic acids are utilized as a growth carbon source for the bacteria of the invention in step (2). An organic acid can be used as a growth carbon source for the bacterium of the present invention without lowering the 4HBA production rate and 4HBA yield. The organic acid concentration of the aqueous solution prepared in step (1) is, for example, 1 to 10,000 mg/L, preferably 50 to 2,000 mg/L. When the aqueous solution prepared in step (1) contains two or more organic acids, the organic acid concentration means the total concentration of the two or more organic acids.

<Step (2)>
In the step (2), bacteria deficient in PobA enzyme expression are allowed to act on the aqueous solution prepared in the step (1), the bacteria metabolize the ferulic acid as a carbon source for growth, and the bacteria convert the coumaric acid into 4 - is a step of converting to hydroxybenzoic acid. The step (2) will be described below.

The PobA enzyme is an enzyme also called 4-hydroxybenzoate hydroxylase, and is one of the benzoate-degrading enzymes. Naturally occurring bacteria include those that possess the PobA enzyme and those that do not. A bacterium deficient in PobA enzyme expression can be obtained by deficient PobA enzyme expression in the bacterium of interest. The target bacterium to be deficient in PobA enzyme expression refers to a bacterium that originally possesses the PobA enzyme. It refers to inactivated bacteria. Preferably, the PobA enzyme expression-deficient bacterium is a bacterium that originally possesses the PobA enzyme. It refers to functionally inactivated bacteria. Bacteria deficient in PobA enzyme expression include, for example, disruption of the pobA gene (e.g., insertion of a disruption cassette into the pobA gene, replacement of the pobA gene with the disruption cassette, deletion (deletion) of the pobA gene, etc.), pobA gene Bacteria in which the PobA enzyme has been functionally inactivated by introducing mutations into , blocking the control region of the pobA gene, or the like. Bacteria in which the PobA enzyme is functionally inactivated include, for example, bacteria in which the activity of the PobA enzyme is reduced or eliminated compared to the bacterium (e.g., wild-type bacterium) before the PobA enzyme expression is deficient; Bacteria in which the level of expression of the PobA enzyme is reduced compared to bacteria (for example, wild-type bacteria) before the expression of the PobA enzyme is deficient; bacteria that do not express the PobA enzyme; On the other hand, some bacteria do not originally possess the PobA enzyme, but in the case of such bacteria, the enzymatic metabolic conversion pathway of coumaric acid and ferulic acid does not exist in the first place, and the desired 4-hydroxybenzoic acid cannot be produced. I don't like to use it because it's difficult.

Bacteria preferably usable in the present invention include Pseudomonas, Rhodococcus, Enterobacter, Bacillus, Mycoplana, Xanthomonas, Achromobacter, etc. Most preferably, Pseudomonas bacterium.

PobA enzymes originally possessed by Pseudomonas bacteria include, for example, the following proteins (a) and (b).
(a) a protein comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2; Preferably 80% or more, more preferably 90% or more (e.g., 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more ) comprising or consisting of an amino acid sequence having the sequence identity of ), which protein has PobA enzymatic activity

The amino acid sequence set forth in SEQ ID NO: 2 is the amino acid sequence (Accession No. Q88H28) of the PobA enzyme derived from Pseudomonas putida strain KT2440.

Sequence identity of amino acid sequences is calculated using homology search software such as BLAST (J. Mol. Biol., 215, 403 (1990)) and FASTA (Methods in Enzymology, 183, 63 (1990)). can be done.

Examples of the gene (pobA gene) encoding the PobA enzyme originally possessed by Pseudomonas bacteria include the following DNAs (c) and (d).
(c) DNA containing or consisting of the nucleotide sequence set forth in SEQ ID NO: 1
(d) A DNA that can hybridize under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1 and that encodes a protein having PobA enzymatic activity.

The nucleotide sequence set forth in SEQ ID NO: 1 is the nucleotide sequence of the pobA gene (Accession No. AAN69138) derived from Pseudomonas putida strain KT2440.

The DNA of (d) is obtained, for example, by colony hybridization, plaque hybridization, Southern blot hybridization, etc., using the DNA consisting of the base sequence of SEQ ID NO: 1 or a fragment thereof as a probe. be able to. More specifically, DNA that hybridizes under stringent conditions is filtered at 65° C. in the presence of 0.7-1.0 M NaCl using filters on which DNA from colonies or plaques has been immobilized. After hybridization, filter was washed at 65°C in 0.1-2x SSC solution (1x SSC solution has 150mM NaCl and 15mM sodium citrate) can do. Hybridization can be performed using known methods such as those described in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory (2001). The higher the temperature or the lower the salt concentration, the higher the stringency and the more homologous (sequence identity) polynucleotides can be isolated.

In addition, the pobA gene is 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 80% or more of the base sequence of SEQ ID NO: 1, as long as it encodes a protein having PobA enzymatic activity. has 90% or more (e.g., 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity It may be a DNA containing the sequence or consisting of the base sequence. For example, the pobA gene may be DNA in which a codon is replaced with its equivalent codon based on codon degeneracy.

The sequence identity of base sequences is calculated using homology search software such as BLAST (J. Mol. Biol., 215, 403 (1990)) and FASTA (Methods in Enzymology, 183, 63 (1990)). can be done.

The present invention is characterized by utilizing bacteria deficient in PobA enzyme expression. The bacterium deficient in PobA enzyme expression is preferably a Pseudomonas bacterium deficient in PobA enzyme expression. A Pseudomonas bacterium deficient in PobA enzyme expression refers to a Pseudomonas bacterium that originally possesses the PobA enzyme, in which the PobA enzyme has been functionally inactivated. Pseudomonas bacteria deficient in PobA enzyme expression include, for example, disruption of the pobA gene (e.g., insertion of a disruption cassette into the pobA gene, replacement of the pobA gene with the disruption cassette, deletion (deletion) of the pobA gene, etc.), Pseudomonas bacterium in which the PobA enzyme is functionally inactivated by introducing a mutation into the pobA gene, blocking the control region of the pobA gene, or the like. A Pseudomonas bacterium in which the PobA enzyme is functionally inactivated is, for example, a Pseudomonas bacterium (for example, a wild-type bacterium) before deficient expression of the PobA enzyme, in which the activity of the PobA enzyme is reduced or eliminated. Pseudomonas bacterium in which the expression level of the PobA enzyme is reduced compared to the Pseudomonas bacterium (e.g., wild-type bacterium) before the expression of the PobA enzyme is deleted; Pseudomonas bacterium that does not express the PobA enzyme bacteria and the like.

Pseudomonas bacteria include Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas agarici, Pseudomonas alcarigenes, Pseudomonas alkaline Phila (Pseudomonas alcaliphila) can be exemplified. , preferably Pseudomonas putida.

Known methods for deficient expression of the PobA enzyme in target bacteria include methods of disrupting the pobA gene by homologous recombination. In addition, methods such as introduction of mutations into the pobA gene and inactivation of the pobA gene are also known. Genetic manipulations such as phage transduction, homologous recombination, plasmid vector insertion, and the like can be used to abolish PobA enzyme expression in the bacterium of interest. Methods using homologous recombination include gene knockout for gene disruption and gene knockin for mutation introduction.

In one embodiment, a method of disrupting the pobA gene of a wild-type bacterium by homologous recombination is used as a method of deficient PobA enzyme expression in a target bacterium. Disruption of the pobA gene may be insertion of a disruption cassette into the pobA gene by homologous recombination, replacement of the pobA gene with the disruption cassette by homologous recombination, or pobA gene by homologous recombination. may be a deletion of As a vector, for example, a plasmid such as pK18mobsacB (GenBank: FJ437239.1) (Gene, 145, 69-73 (1994)) can be used. Methods for introducing vectors into wild-type bacteria include, for example, electroporation, calcium phosphate coprecipitation, lipofection, microinjection, lithium acetate, and the like. The pobA gene disruption cassette may contain a selectable marker gene such as a drug resistance gene (eg, kanamycin resistance gene, hygromycin resistance gene, zeocin resistance gene, chloramphenicol resistance gene, etc.). A selectable marker gene may be included in the vector. Transformed cells in which homologous recombination has occurred can be obtained by selecting cells with drugs corresponding to selectable marker genes (eg, kanamycin, hygromycin, zeocin, chloramphenicol, etc.). The occurrence of insertion into the target gene or substitution or deletion of the target gene by homologous recombination in the obtained transformed cells can be confirmed by PCR using primers capable of amplifying the target gene.

By culturing the PobA enzyme expression-deficient bacterium in the aqueous solution prepared in step (1), the PobA enzyme expression-deficient bacterium can act on the aqueous solution prepared in step (1). The culture temperature is, for example, 15 to 40° C., preferably 20 to 30° C., and the culture period is, for example, 1 hour to 10 days, preferably 4 to 48 hours. Cultivation is, for example, aerobic culture, and aerobic culture can be performed by, for example, shaking the culture solution or blowing air or oxygen gas into the culture solution. The pH of the aqueous solution when the PobA enzyme expression-deficient bacterium is allowed to act is preferably in the range of pH 4 to 9, and more preferably in the range of pH 5 to 7 from the viewpoint of conversion efficiency to 4HBA.

When the PobA enzyme expression-deficient bacterium is allowed to act on the aqueous solution prepared in step (1), the PobA enzyme expression-deficient bacterium proliferates while metabolizing ferulic acid in the aqueous solution as a carbon source for growth. of coumaric acid is converted to 4-hydroxybenzoic acid (4HBA). In addition, since bacteria originally possess the PobA enzyme, the converted 4HBA is further degraded or converted, but bacteria lacking PobA enzyme expression do not further degrade or convert 4HBA, so 4HBA is cultured. Accumulates in liquids. When the aqueous solution prepared in step (1) contains carbon sources such as organic acids and amino acids, and nitrogen sources, PobA enzyme expression-deficient bacteria grow while consuming and decomposing these. That is, in the present invention, impurity components other than 4HBA in the culture medium are removed by consuming carbon sources and nitrogen sources other than 4HBA by bacteria deficient in PobA enzyme expression, thereby removing 4HBA from the culture medium. Purification can be simplified.

　4HBA can be further purified from an aqueous solution in which bacteria deficient in PobA enzyme expression are allowed to act. For purification, bacteria deficient in PobA enzyme expression are isolated, the separated filtrate is passed through an adsorption column, and the adsorbed fraction is recovered, thereby further increasing the purification purity of HBA. As the adsorption column, there are a hydrophobic adsorption column, an ion exchange column and the like, and a hydrophobic adsorption column is preferable.

4HBA in the present invention is a general term for free 4HBA, salts and esters of 4HBA, and the 4HBA obtained in the present invention may be in any of these forms or a mixture thereof. 4HBA ester is also called paraben, and preferred specific examples include methyl ester (methylparaben), ethyl ester (ethylparaben), propyl ester (propylparaben), isopropyl ester (isopropylparaben), butyl ester (butylparaben), Isobutyl ester (isobutylparaben) may be mentioned.

The 4HBA obtained in the present invention can be used as a raw material for liquid crystal polymers or a precursor for chemical synthesis, like 4HBA obtained from other raw materials. For example, ACS Catal. 2016, 6, 9, 6141-6145 Production of Terephthalic Acid from Lignin-Based Phenolic Acids by a Cascade Fixed-Bed Process It can be used as a raw material for terephthalic acid, which is a raw material for PET.

The present invention will be specifically described below with reference to examples. However, the present invention is not limited to these.

(Reference Example 1) Measurement of Sugar Concentration The concentrations of glucose and xylose contained in the bagasse alkaline extract were quantified by comparison with standard products under the HPLC conditions shown below.
Column: Luna NH ₂ (manufactured by Phenomenex)
Mobile phase: MilliQ: acetonitrile = 25:75 (flow rate 0.6 mL/min)
Reaction liquid: None Detection method: RI (differential refractive index)
Temperature: 30°C.

(Reference Example 2) Analysis of Aromatic Compounds Aromatic compounds contained in the bagasse alkaline extract were quantified by comparison with standard samples under the HPLC conditions shown below. Each analysis sample was centrifuged at 3500 G for 10 minutes, and the supernatant components were subjected to the following analysis.
Column: Synergi HydroRP 4.6 mm × 250 mm (manufactured by Phenomenex)
Mobile phase: Acetonitrile-0.1% H ₃ PO ₄ (flow rate 1.0 mL/min) Detection method: UV (283 nm)
Temperature: 40°C.

Acetic acid and formic acid contained in the bagasse alkaline extract were quantified by comparison with standard products under the HPLC conditions shown below. Each analysis sample was centrifuged at 3500 G for 10 minutes, and the supernatant components were subjected to the following analysis.
Column: Series of Shim-Pack and Shim-Pack SCR101H (manufactured by Shimadzu Corporation) Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL/min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bis-Tris, 0.1 mM EDTA·2Na (flow rate 0.8 mL/min)
Detection method: electrical conductivity Temperature: 45°C.

(Example 1) Alkali treatment of lignin-containing biomass Bagasse was prepared as lignin-containing biomass. 85 g of 14% NaOH solution and 1787 g of RO water were added to 128 g of bagasse (moisture content of 22%, equivalent to 100 g of dry bagasse) and mixed. That is, 12 g of NaOH was added to 100 g of dry bagasse, and the amount of NaOH added per 1 g of dry bagasse was 120 mg. The final concentration of bagasse solids was 5 wt %. After that, alkali treatment was performed at 90° C. for 2 hours in an autoclave. The bagasse after alkali treatment was subjected to 1 mm screen filtration, and then the filtrate was subjected to 212 μm screen filtration to recover a total of about 1500 g of alkali treated liquid. The pH at this time was 12.7. 1016 g of the alkaline treatment liquid was used for pH adjustment. While monitoring the pH change with a pH sensor, 19 g of 6M HCl was added to adjust the pH to 5.2. After that, the pH-adjusted liquid was centrifuged at 8500 G for 6 minutes, and the supernatant was used as a bagasse alkaline extract.

Table 1 shows the component composition of the bagasse alkali extract.

(Example 2) Preparation of a Pseudomonas bacterium lacking PobA enzyme expression (KT2440 strain ΔPobA strain) In the genome sequence of Pseudomonas putida strain KT2440, the base sequence of 1000 bp upstream from the 5' end of the start codon of the pobA gene and the pobA gene digested with BamHI and EcoRI, and ligated with pK18mobsacB (obtained from the National Institute of Genetics) similarly digested with BamHI and EcoRI. Then, a homologous recombination vector (pK18ΔpobA) for deleting the pobA gene was constructed. Pseudomonas putida (P. putida) strain KT2440 was inoculated into 10 mL of LB liquid medium and cultured with shaking at 30° C. for 16 hours. A portion of the culture was inoculated into 5 mL of fresh LB liquid medium and cultured with shaking at 30° C. until OD ₆₀₀ reached 0.3-0.5. Cells were collected from the resulting culture medium by centrifugation (6,000×g, 5 min, 4° C.) and washed with a cooled 10% Glycerol solution. The washing operation was repeated 3 times. The washed cells were collected by centrifugation and suspended in 0.1 mL of chilled 10% Glycerol solution. Cell suspension and 1 μg of pK18ΔpobA were mixed and subjected to electroporation (12.5 KV). The mixture was mixed with 1 mL of SOC medium and cultured with shaking at 30° C. for 1 hour. Cells were harvested by centrifugation and suspended in 0.1 mL of saline. The suspension was smeared on an LB agar medium containing 25 mg/L kanamycin and statically cultured overnight at 30°C. The formed kanamycin-resistant colonies were inoculated into 5 mL of LB liquid medium containing 10% sucrose and cultured with shaking at 30° C. for 16 hours. A portion of the obtained culture solution was inoculated into 5 mL of LB liquid medium containing 10% sucrose, and shake culture was performed at 30° C. for 16 hours, which was repeated twice. A portion of the obtained culture solution was smeared on an LB agar medium and statically cultured at 30° C. overnight. The genomic DNA of the formed colony is extracted by a conventional method, and is used as a template. pobA by PCR using a primer set consisting of a primer) and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 4 (a primer that anneals to a region located approximately 1.4 kbase upstream from the 5′ end of the pobA gene). Gene deletion was confirmed. This bacterium was used in the following examples as Pseudomonas bacterium (KT2440 strain ΔPobA strain) deficient in PobA enzyme expression.

(Example 3) Preculture of Pseudomonas bacterium (KT2440 strain ΔPobA strain) deficient in PobA enzyme expression KT2440 strain ΔPobA strain was statically cultured overnight at 30°C on an LB agar medium. A colony formed on the agar medium was inoculated into 10 mL of an LB liquid medium and cultured with shaking at 30° C. for 16 hours. 1 mL of a cell suspension (suspended in physiological saline) having an OD ₆₀₀ of 5.0 was prepared from the obtained culture medium and used as an inoculum.

(Example 4) An example of the process of converting Pseudomonas bacteria into 4HBA by acting on an alkaline extract solution. 1 mL of 342.0 g/L Na ₂ HPO ₄ .12H ₂ O, 60.0 g/L KH ₂ PO ₄ , 20.0 g/L NH ₄ Cl) was added, and 100×Metal (49.0 g/L MgSO ₄ . 7H ₂ O, 0.5 g/L FeSO ₄ .7H ₂ O, 1.5 g/L CaCl ₂ .2H ₂ O) was added in 0.1 mL to prepare a total of 10.1 mL of extract. 0.1 mL of the inoculum obtained in Example 3 was added thereto, and cultured with shaking at 30°C. The _OD600 at the start of culture was 0, and the _OD600 24 hours after the start of culture was 1.2. Table 2 shows a comparison of components at the start of culture and 24 hours after the start of culture.

By comparing the components at the start of culture and 24 hours after the start of culture, it was confirmed that coumaric acid was consumed and 4-hydroxybenzoic acid was accumulated. The conversion yield from coumaric acid to 4-hydroxybenzoic acid was 100.4 mol %. On the other hand, other aromatic compounds such as ferulic acid and vanillic acid and organic acids such as acetic acid, formic acid and lactic acid decreased, confirming that they were used as carbon sources for the growth of Pseudomonas bacteria.

(Example 5) Another example of the process of converting Pseudomonas bacteria into 4HBA by acting on an alkaline extract (in the presence of glucose)
After adjusting the extract of the composition in Table 1 _to pH 7, 9 mL was used for 4HBA _conversion , plus 10 x MM (342.0 g/L _{Na2HPO4.12H2O} , 60.0 g/L _KH2PO4 , 20.0 g/L NH ₄ Cl) was added to 1 mL, and 100×Metal (49.0 g/L MgSO ₄ .7H ₂ O, 0.5 g/L FeSO ₄ .7H ₂ O, 1.5 g/L CaCl ₂ . 2H ₂ O) was added to prepare a total of 10.1 mL of extract. Furthermore, glucose was added to a final concentration of 10 g/L. 0.1 mL of the inoculum obtained in Example 3 was added thereto, and cultured with shaking at 30°C. The _OD600 at the start of culture was 0, and the _OD600 24 hours after the start of culture was 2.5, confirming better cell growth than in Example 4. Table 3 shows a comparison of components at the start of culture and 24 hours after the start of culture.

In Example 5, as in Example 4, it was confirmed that coumaric acid was consumed and 4-hydroxybenzoic acid was accumulated. The conversion yield from coumaric acid to 4-hydroxybenzoic acid was 88.2 mol %, lower than that of Example 4. In addition, it was confirmed that other aromatic compounds such as ferulic acid and vanillic acid and organic acids such as acetic acid, formic acid and lactic acid were reduced but remained unused compared to Example 4. That is, it was confirmed that less than 5 g/L of glucose, or even no addition of glucose, is preferable because impurities after culture can be reduced and conversion efficiency of 4-hydroxybenzoic acid can be increased.

(Example 6) Another example of the process of converting Pseudomonas bacteria into 4HBA by acting on an alkaline extract (effect of pH)
After adjusting the extract having the composition shown in Table 1 to five conditions of pH 4.5, pH 5.0, pH 6.0, pH 7.0, and pH 8.0, 9 mL was used for 4HBA conversion, and 10 × MM ( 1 mL of 342.0 g/L Na ₂ HPO ₄ .12H ₂ O, 60.0 g/L KH ₂ PO ₄ , 20.0 g/L NH ₄ Cl) was added, and 100×Metal (49.0 g/L MgSO ₄ . 7H ₂ O, 0.5 g/L FeSO ₄ .7H ₂ O, 1.5 g/L CaCl ₂ .2H ₂ O) was added in 0.1 mL to prepare a total of 10.1 mL of extract. 0.1 mL of the inoculum obtained in Example 3 was added thereto, and cultured with shaking at 30°C. Table 4 shows the OD ₆₀₀ after 24 hours from the start of the culture, with the OD ₆₀₀ at the start of the culture set to 0. It was confirmed that the pH range of pH 5 to 7 is preferable for the pH condition in the step of converting Pseudomonas bacterium into 4HBA by acting on the alkaline extract.

(Comparative Example 1) Step of converting Pseudomonas bacteria to model extract to convert to 4HBA As a model extract, reagents for each aromatic compound were mixed to prepare a model extract having the composition shown in Table 5. A culture test was performed in the same manner as in Example 4, except that the extract was changed to the model extract in Table 5. Pseudomonas bacteria could grow only to OD ₆₀₀ =0.2 24 days after the start of culture.

Possibility of industrial use

Using biomass as a raw material, it is possible to produce high-purity 4-hydroxybenzoic acid with a small number of purification steps.

Claims

A method for producing 4-hydroxybenzoic acid, comprising the following steps (1) and (2).
Step (1): Preparing an aqueous solution containing coumaric acid and ferulic acid Step (2): Bacteria deficient in PobA enzyme expression are allowed to act on the aqueous solution prepared in Step (1), and the bacteria produce ferulic acid. as a growing carbon source, and converting the coumaric acid to 4-hydroxybenzoic acid by the bacterium
The method for producing 4-hydroxybenzoic acid according to claim 1, wherein the step (1) is a step of subjecting lignin-containing biomass to alkali treatment to obtain an extract containing coumaric acid and ferulic acid as the aqueous solution.
The method for producing 4-hydroxybenzoic acid according to claim 1 or 2, wherein the aqueous solution prepared in step (1) has a sugar concentration of less than 5 g/L.
4. The method according to any one of claims 1 to 3, wherein the aqueous solution prepared in step (1) contains one or more organic acids selected from the group consisting of acetic acid, formic acid and lactic acid. A method for producing 4-hydroxybenzoic acid.
The method for producing 4-hydroxybenzoic acid according to any one of claims 1 to 4, wherein the bacterium used in the step (2) is Pseudomonas bacterium.
The method for producing 4-hydroxybenzoic acid according to any one of claims 1 to 5, wherein in the step (2), the bacteria are allowed to act on the aqueous solution under pH 5-7 conditions.