Method for producing phenols

Abstract

Provided is a method for producing phenols that can be used in the production of phenols from biomass, said method comprising a step for culturing, in a liquid mixture of a culture solution and an organic solvent, a microorganism having the ability to produce phenols, wherein the Hansen solubility parameter (HSP) distance between the organic solvent and water is not less than 38.0, and the HSP distance between the organic solvent and phenol is not more than 14.0.

Description

Method for producing phenols

[0001] The present disclosure relates to a method for producing phenols. More specifically, it relates to a method for producing phenols using microorganisms having the ability to produce phenols.

[0002] In the field of chemical production, a conversion from petroleum-derived raw materials to biomass-derived raw materials is being attempted. Patent Document 1 discloses a technique for fermentatively producing phenol from glucose using a microorganism with a modified sugar metabolism pathway.

[0003] Patent Document 2 also discloses a technique for producing phenol by a bioprocess using a phenol-producing bacterium. The technique disclosed in Patent Document 2 uses a phenol extractant having an active ingredient of a hydrocarbon with a LogP of 5 or more, being liquid at 25°C, and having at least one hydrogen atom substituted with a hydroxyl group, to extract phenol from a culture of a phenol-producing bacterium. This phenol extractant has high phenol extraction efficiency, low toxicity to phenol-producing bacteria, and good separation between the organic phase and the aqueous phase.

[0004] Patent Document 2 describes reusing the aqueous phase (wastewater) remaining after recovering phenol from a culture of a phenol-producing bacterium using a phenol extractant for culturing the phenol-producing bacterium, but does not assume a two-phase culture at all, and does not describe reusing the aqueous phase (wastewater) in a method for producing phenols in a two-phase culture. Further, the technique disclosed in Patent Document 2 includes a step of recovering an organic phase from a mixture of a culture of a phenol-producing bacterium and a phenol extractant, but does not perform separation and recovery of the cells and resupply to the culture system.

[0005] Re-Patent No. 2017 - 033965, JP-A-2017-105720

[0006] The main object of the present disclosure is to provide a technique that can be used for producing phenols from biomass.

[0007] To address the above issues, this disclosure provides the following [1]-

[11] : [1] A method for producing phenols, comprising step 1 of culturing a microorganism having phenol-producing ability in a mixture of culture medium and an organic solvent, wherein the Hansen solubility parameter (HSP) distance of the organic solvent with water is 38.0 or greater, and the HSP distance with phenol is 14.0 or less. [2] The method according to [1], wherein the organic solvent is an ester compound. [3] The method for producing phenols according to [2], wherein the organic solvent is a compound that can be used as a plasticizer. [4] The method according to [3], wherein the plasticizer is a plasticizer for vinyl chloride resins. [5] The method according to [2], wherein the organic solvent is at least one selected from the group consisting of trialkyl phosphate, diadipate, dibutyrate, dibacate, and diester acetate. [6] The method for producing organic solvent according to [5], wherein the organic solvent is at least one selected from the group consisting of tributyl phosphate, tris(2-ethylhexyl) phosphate, triphenyl phosphate, 2-ethylhexyldiphenyl phosphate, trityl phosphate, triamyl phosphate, tri-o-cresyl phosphate, cresyldiphenyl phosphate, diisononyl adipate, bis(2-ethylhexyl) adipate, dioctyl adipate, diisodecyl adipate, 2,2,4-trimethylpentane-1,3-diyldiisobutyrate, ethylene glycol dibutyrate, diisobutyl adipate, bis(2-ethylhexyl) adipate, dibutyl sebacate, and 2-ethylhexyl acetate. [7] The method for producing organic solvent according to any one of [1] to [6], wherein the phenols are at least one selected from the group consisting of phenol, catechol, cresol, xylenol, 4-hydroxybenzoic acid, and salicylic acid. [8] A method for producing a culture according to any one of [1] to [7], further comprising: step 2 of separating at least an organic solvent from the culture obtained in step 1; and step 3 of recovering phenols from the separated organic solvent. [9] A method for producing a culture according to [8], further comprising the step of separating bacterial cells from the culture obtained in step 1, wherein the separated bacterial cells are again used for culturing in step 1.

[10] A method for producing phenols according to [8], further comprising the step of separating a culture medium from the culture obtained in step 1, wherein the separated culture medium is again used for culturing in step 1.

[11] A method for producing phenols according to

[10] , wherein the separated culture medium is subjected to treatment with activated carbon and / or ion exchange resin, or is untreated and again used for culturing in step 1.

[0008] To address the above issues, this disclosure provides, in another aspect, the following [1A]-[18A]: [1A] A method for producing phenols, comprising step 1A of culturing a microorganism having phenol-producing ability in a mixture of a culture medium (hereinafter also referred to as the "aqueous phase") and an organic solvent (hereinafter also referred to as the "organic phase"), wherein the Hansen solubility parameter (HSP) distance of the organic solvent with water is 38.0 or greater, and the HSP distance with phenol is 14.0 or less. [2A] The method for producing phenols according to [1A], wherein the organic solvent is a compound that can be used as a plasticizer, particularly a plasticizer for vinyl chloride resins. [3A] The method for producing phenols according to [2A], wherein the organic solvent is an ester compound. [4A] The method for producing phenols according to [3A], wherein the organic solvent is at least one selected from the group consisting of trialkyl phosphate, diadipate, and dibutyrate. [5A] The method for producing organic solvent according to [1A] or [4A], wherein the organic solvent is at least one selected from the group consisting of tributyl phosphate, tris(2-ethylhexyl) phosphate, triphenyl phosphate, 2-ethylhexyldiphenyl phosphate, trityl phosphate, triamyl phosphate, tri-o-cresyl phosphate, cresyldiphenyl phosphate, diisononyl adipate, bis(2-ethylhexyl) adipate, dioctyl adipate, diisodecyl adipate, 2,2,4-trimethylpentane-1,3-diyldiisobutyrate, and ethylene glycol dibutyrate. [6A] The method for producing organic solvent according to any one of [1A] to [5A], wherein the phenol is at least one selected from the group consisting of phenol, catechol, cresol, xylenol, 4-hydroxybenzoic acid, and salicylic acid. [7A] A method for producing the mixture according to any one of [1A] to [6A], further comprising: (1) separating the bacterial cells first and then separating the aqueous phase and the organic phase; (2) separating the organic phase first and then separating the aqueous phase and the bacterial cells; or (3) a step 2A in which the organic phase, aqueous phase and the bacterial cells are separated; and a step 3A in which the phenols are recovered from the organic phase.[8A] The method for producing phenols according to any one of [1A] to [7A], wherein the microorganism having the ability to produce phenols is a recombinant having a 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase gene, a transketolase gene, a colismyate mutase / prephenate dehydrogenase gene, a phosphoenolpyruvate synthase gene, and a tyrosine phenol lyase gene that has been introduced in an expressible manner. [9A] The method for producing phenols, wherein the microorganism having phenol-producing ability is a mutant strain having a gene mutation introduced into a parent strain, with a genetically modified parent strain having an expressibly introduced 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase gene, transketolase gene, colismyate mutase / prephenate dehydrogenase gene, phosphoenolpyruvate synthase gene and tyrosine phenol lyase gene, wherein the mutant strain shows improved growth compared to the parent strain when cultured in a phenol-containing culture medium and exhibits enhanced phenol-producing ability compared to the parent strain when used in biomass fermentation. [10A] The method for producing phenols according to [8A] or [9A], wherein the genetically modified organism is Escherichia coli or yeast. [11A] A method for producing phenols comprising: step 1A of culturing a microorganism capable of producing phenols in a mixture of culture medium and an organic solvent; step 2A of separating the mixture by (1) first separating the microbial cells and then separating the aqueous phase and the organic phase, (2) first separating the organic phase and then separating the aqueous phase and the microbial cells, or (3) separating the organic phase, the aqueous phase and the microbial cells; and step 3A of recovering the phenols from the organic phase, wherein the organic solvent has a Hansen solubility parameter (HSP) distance of 38.0 or more with water and an HSP distance of 14.0 or less with phenol. [12A] The use according to [11A], wherein the organic solvent is at least one selected from the group consisting of trialkyl phosphate, diadipate, and dibutyrate.[13A] The use according to [12A], wherein the organic solvent is at least one selected from the group consisting of tributyl phosphate, tris(2-ethylhexyl) phosphate, triphenyl phosphate, 2-ethylhexyldiphenyl phosphate, trityl phosphate, triamyl phosphate, tri-o-cresyl phosphate, cresyldiphenyl phosphate, diisononyl adipate, bis(2-ethylhexyl) adipate, dioctyl adipate, diisodecyl adipate, 2,2,4-trimethylpentane-1,3-diyldiisobutyrate, and ethylene glycol dibutyrate. [14A] The use according to any one of [11A]-[13A], wherein the phenol is at least one selected from the group consisting of phenol, catechol, cresol, xylenol, 4-hydroxybenzoic acid, and salicylic acid. [15A] An extractant for phenols from a culture medium of a microorganism having phenol-producing ability, comprising an organic solvent having a Hansen solubility parameter (HSP) distance of 38.0 or more with water and an HSP distance of 14.0 or less with phenol. [16A] The extractant according to [15A], wherein the organic solvent is at least one selected from the group consisting of trialkyl phosphate, diadipate, and dibutyrate. [17A] The extractant according to [16A], wherein the organic solvent is at least one selected from the group consisting of tributyl phosphate, tris(2-ethylhexyl) phosphate, triphenyl phosphate, 2-ethylhexyldiphenyl phosphate, trityl phosphate, triamyl phosphate, tri-o-cresyl phosphate, cresyldiphenyl phosphate, diisononyl adipate, bis(2-ethylhexyl) adipate, dioctyl adipate, diisodecyl adipate, 2,2,4-trimethylpentane-1,3-diyldiisobutyrate, and ethylene glycol dibutyrate. [18A] The extractant according to any one of [15A] to [17A], wherein the phenol is at least one selected from the group consisting of phenol, catechol, cresol, xylenol, 4-hydroxybenzoic acid, and salicylic acid.

[0009] To address the above issues, this disclosure provides, in another aspect, the following [1B]-[11B]: [1B] A method for producing phenols, comprising: step 1B of culturing a microorganism having the ability to produce phenols in a mixture of a culture medium and an organic solvent; step 2B of separating at least the culture medium from the culture obtained in step 1B; and step 3B of using the culture medium separated in step 2B again for culturing in step 1B. [2B] The method for producing phenols according to [1B], further comprising the step of extracting phenols from the organic solvent separated from the culture. [3B] The manufacturing method according to [2B], wherein in step 2B, (1) bacterial cells are separated from the culture obtained in step 1B, and then the culture solution and an organic solvent containing phenols are separated, (2) the organic solvent containing phenols is separated from the culture obtained in step 1B, and then the culture solution and bacterial cells are separated, (3) the culture solution is separated from the culture obtained in step 1B, and then the organic solvent containing phenols and bacterial cells are separated, or (4) the culture solution, the organic solvent containing phenols, and bacterial cells are simultaneously separated from the culture obtained in step 1B. [4B] The manufacturing method according to any one of [1B] to [3B], wherein the culture solution separated in step 2B is subjected to treatment with activated carbon and / or ion exchange resin, or is subjected to cultivation again in step 1B without treatment. [5B] The manufacturing method according to [3B] or [4B], wherein the organic solvent after phenol recovery in step 2B is subjected to cultivation again in step 1B. [6B] The manufacturing method according to any one of [1B] to [5B], wherein the organic solvent is at least one selected from the group consisting of trialkyl phosphates, diadipates, and diesters of butyrate.[7B] The method for producing an organic solvent according to [6B], wherein the organic solvent is at least one selected from the group consisting of tributyl phosphate, tris(2-ethylhexyl) phosphate, triphenyl phosphate, 2-ethylhexyldiphenyl phosphate, trityl phosphate, triamyl phosphate, tri-o-cresyl phosphate, cresyldiphenyl phosphate, diisononyl adipate, bis(2-ethylhexyl) adipate, dioctyl adipate, diisodecyl adipate, 2,2,4-trimethylpentane-1,3-diyldiisobutyrate, and ethylene glycol dibutyrate. [8B] The method for producing an organic solvent according to any one of [1B] to [7B], wherein the phenol is at least one selected from the group consisting of phenol, catechol, cresol, xylenol, 4-hydroxybenzoic acid, and salicylic acid. [9B] The method for producing phenols according to any one of [1B] to [8B], wherein the microorganism having the ability to produce phenols is a recombinant having a 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase gene, a transketolase gene, a colismyate mutase / prephenate dehydrogenase gene, a phosphoenolpyruvate synthase gene, and a tyrosine phenol lyase gene that has been introduced in an expressible manner. [10B] The method for producing phenols according to any one of [1B] to [8B], wherein the microorganism having the ability to produce phenols is a mutant strain having a gene mutation introduced into a parent strain, with a genetically modified parent strain having an expressibly introduced 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase gene, transketolase gene, colismic acid mutase / prephenate dehydrogenase gene, phosphoenolpyruvate synthase gene and tyrosine phenol lyase gene, wherein the mutant strain shows improved growth than the parent strain when cultured in a phenol-containing culture medium and exhibits enhanced phenol production ability than the parent strain when used in biomass fermentation. [11B] The method for producing phenols according to [9B] or [10B], wherein the genetically modified organism is Escherichia coli or yeast.

[0010] To address the above issues, this disclosure provides, in another aspect, the following [1C]-[9C]. [1C] A method for producing phenols, comprising: step 1C of culturing a microorganism capable of producing phenols in a mixture of a culture medium and an organic solvent; and step 2C of separating at least microbial cells from the culture obtained in step 1C, wherein the microbial cells separated in step 2C are again subjected to the culturing in step 1C, and the Hansen solubility parameter (HSP) distance of the organic solvent to water is 40.0 or more and 45.0 or less, and the HSP distance to phenol is 12.0 or more and 14.0 or less. [2C] The method for producing phenols according to [1C], further comprising the step of extracting phenols from the organic solvent separated from the culture. [3C] The method for producing the organic solvent according to [1C] or [2C], wherein the organic solvent is one or more selected from the group consisting of 2,2,4-trimethylpentane-1,3-diyldiisobutyrate (TXIB), tris(2-ethylhexyl) phosphate (TEP), bis(2-ethylhexyl) adipate (DEHA), diisodecyl adipate (DIDA), and isostearyl alcohol. [4C] The method for producing the organic solvent according to any one of [1C] to [3C], wherein the phenol is at least one selected from the group consisting of phenol, catechol, cresol, xylenol, 4-hydroxybenzoic acid, and salicylic acid. [5C] The method for producing phenols according to any one of [1C] to [4C], wherein the microorganism having the ability to produce phenols is a recombinant having an expressibly introduced 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase gene, a transketolase gene, a colismyate mutase / prephenate dehydrogenase gene, a phosphoenolpyruvate synthase gene, and a tyrosine phenol lyase gene.[6C] The method for producing phenols, wherein the microorganism having phenol-producing ability is a mutant strain having a gene mutation introduced into a parent strain, with a genetically modified parent strain having an expressibly introduced 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase gene, transketolase gene, colismic acid mutase / prephenate dehydrogenase gene, phosphoenolpyruvate synthase gene and tyrosine phenol lyase gene, wherein the mutant strain shows improved growth compared to the parent strain when cultured in a phenol-containing medium and exhibits enhanced phenol-producing ability compared to the parent strain when used in biomass fermentation, as described in any one of [1C] to [5C]. [7C] The method for producing [5C] or [6C], wherein the genetically modified organism is Escherichia coli or yeast. [8C] A method for producing phenols, comprising: step 1C of culturing a microorganism capable of producing phenols in a mixture of culture medium and an organic solvent; step 2C of separating the culture obtained in step 1C into an aqueous phase, an organic phase, and microbial cells; and step 3C of recovering phenols from the organic phase, wherein the microbial cells separated in step 2C are again subjected to the culturing in step 1C, wherein the organic solvent is one or more selected from the group consisting of 2,2,4-trimethylpentane-1,3-diyldiisobutyrate (TXIB), tris(2-ethylhexyl) phosphate (TEP), bis(2-ethylhexyl) adipate (DEHA), diisodecyl adipate (DIDA), and isostearyl alcohol. [9C] The use according to [8C], wherein the phenols are at least one selected from the group consisting of phenol, catechol, cresol, xylenol, 4-hydroxybenzoic acid, and salicylic acid.

[0011] This disclosure provides a technology that can be used for the production of phenols from biomass.

[0012] This graph shows the results of an investigation into the effect of culture medium recycling on phenol productivity in a flask-based phenol production reaction. It evaluates the phenol production rate when a phenol-producing bacterium (MCP1.1.1 strain) was cultured for the first time in a mixture of culture medium and various organic solvents (TXIB, TEP, triptyline), and the phenol production rate when the phenol-producing bacterium was isolated and recovered from the culture of the first culture and cultured a second time. The phenol production rate is shown as a relative value with the rate during the first culture set to 1.

[0013] The following describes preferred forms for implementing this disclosure. The embodiments described below are merely examples of typical embodiments of this disclosure and should not be interpreted as narrowing the scope of this disclosure.

[0014] The method for producing phenols according to this disclosure includes the following step 1, and optionally includes steps 2 and 3. The culture solution separated in step 2 may be used again for the culture in step 1. The microbial cells separated in step 2 may also be used again for the culture in step 1. Step 1: A step of culturing a microorganism capable of producing phenols in a mixture of culture solution and an organic solvent. Step 2: From the mixture (culture) after step 1, (1) first separate the microbial cells and then separate the aqueous phase and the organic phase, (2) first separate the organic phase and then separate the aqueous phase and the microbial cells, or (3) separate the organic phase, aqueous phase and the microbial cells. Step 3: A step of recovering phenols from the organic phase separated in step 2.

[0015] Step 1: In this step, microorganisms capable of producing phenols are cultured in a mixture of culture medium (hereinafter also referred to as "culture medium") and an organic solvent. The culture is preferably carried out by two-phase culture using a culture carrier containing a culture medium (aqueous phase) and an organic solvent (organic phase). The phenols may be at least one selected from the group consisting of phenol, catechol, cresol, xylenol, 4-hydroxybenzoic acid, and salicylic acid. The phenols are preferably phenols.

[0016] The microorganisms can be any microorganism capable of producing phenols, and examples of wild strains include the following: Escherichia bacteria such as Escherichia coli; Pseudomonas bacteria such as Pseudomonas putida; Corynebacterium bacteria such as Corynebacterium glutamicum; Bacillus bacteria such as Bacillus subtilis; Pichia yeasts such as Pichia pastoris; Saccharomyces yeasts such as Saccharomyces cerevisiae; and Aspergillus filamentous fungi such as Aspergillus oryzae, Aspergillus nidulans, and Aspergillus niger. One species selected from these can be used alone, or two or more species can be used in combination. Furthermore, the microorganisms are not limited to wild strains, and may also include mutant strains obtained by commonly used mutation treatments such as UV irradiation or chemical treatment, or genetically modified organisms induced by genetic methods such as cell fusion or genetic recombination.

[0017] While there are no particular limitations on the host organisms used for creating genetically modified organisms, bacteria such as Escherichia coli, Rhodococcus, Pseudomonas, Corynebacterium, Bacillus, Streptococcus, and Streptomyces are commonly used; yeasts such as Saccharomyces, Candida, Shizosaccharomyces, and Pichia are commonly used; and filamentous fungi such as Aspergillus are commonly used. Among these, using Escherichia coli and yeast is particularly convenient, efficient, and therefore preferable.

[0018] Microorganisms may be genetically modified organisms that have undergone genetic alterations to enhance their phenol production capacity. Examples of such genetic modifications include enhancing the activity of enzymes involved in tyrosine production, enhancing the activity of enzymes involved in the conversion of tyrosine to phenol, disrupting genes encoding enzymes that degrade tyrosine and phenol, and enhancing the expression of transport proteins that expel the products from the cell.

[0019] Enzyme activity enhancement includes increasing gene expression by introducing the gene encoding the enzyme, improving enzyme activity by introducing mutations into the gene, strongly expressing the gene by strengthening the gene's promoter activity, or increasing expression by reducing or inactivating the gene's repressor activity. Gene introduction and other methods can be carried out according to conventionally known molecular biological techniques. Whether or not enzyme activity has been enhanced can be confirmed by measuring activity using cell-free extracts of microorganisms or purified enzymes.

[0020] Genetic modifications to enhance tyrosine production capacity include strengthening the activity of the 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase gene, the transketolase gene, the colismyate mutase / prephenate dehydrogenase gene, and the phosphoenolpyruvate synthase gene.

[0021] 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase is an enzyme gene that produces 7-phospho-2-dehydro-3-deoxyarabinoheptonate and inorganic phosphate from D-erythrose-4-phosphate, phosphoenolpyruvic acid, and water. This enzyme is known to be inhibited by phenylalanine. It is desirable that the 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase used in this disclosure has a mutation introduced to overcome the inhibition by phenylalanine. Known mutations can be used for this purpose, such as the D146N mutation and the F209S mutation in the enzyme from Escherichia coli (uniport id: P0AB91).

[0022] Transketolase is an enzyme that produces erythritol-4-phosphate and xylulose-5-phosphate from fructose-6-phosphate and glyceraldehyde-3-phosphate.

[0023] Chorismic acid mutase / prephenic acid dehydrogenase is an enzyme that produces 3-(4-hydroxyphenyl)pyruvic acid, NADH, and carbon dioxide from chorismic acid and NAD+. This enzyme is known to be inhibited by tyrosine. It is desirable that the chorismic acid mutase / prephenic acid dehydrogenase used in this disclosure has been modified to release the inhibition by tyrosine. Known mutations can be used for this purpose, such as the M53I mutation and the A354V mutation in the enzyme derived from Escherichia coli (uniport id: P07023).

[0024] Phosphoenolpyruvate synthase is an enzyme that produces phosphoenolpyruvate, AMP, and inorganic phosphate from pyruvate, ATP, and water.

[0025] One method of genetic modification to enhance the ability to convert tyrosine to phenol is to enhance the activity of the tyrosine phenol lyase gene. TPL (EC 4.1.99.2) is an enzyme that uses pyridoxal phosphate as a coenzyme to cleave the carbon-carbon bond of tyrosine, producing phenol, pyruvate, and ammonia. Tyrosine phenol lyase is not particularly limited, but examples include enzymes derived from microorganisms belonging to any of the families Enterobacteriaceae, Clostridiaceae, Fusobacteriaceae, Morganellaceae, or Pasteurellaceae. Alternatively, tyrosine phenol lyase may be an enzyme with the above-mentioned ability, derived from a sequence found in environmental metagenomics, etc.

[0026] Microorganisms belonging to the Enterobacteriaceae family may belong to any of the genera Citrobacter, Pantoea, or Klebsiella. Examples of microorganisms belonging to the genus Citrobacter include C. freundii, C. koseri, and C. youngae. Examples of microorganisms belonging to the genus Pantoea include P. ananatis, P. allii, and P. agglomerans. Examples of microorganisms belonging to the genus Klebsiella include K. oxytoca, K. africana, K. quasivariicola, and K. werkmanii.

[0027] Microorganisms belonging to the Clostridiaceae family may belong to either the Clostridium or Tindallia genera. Examples of microorganisms belonging to the Clostridium genus include C. saccharolyticum, C. tetani, C. tetanomorphum, C. malenominatum, C. cochlearium, C. liquoris, C. pascui, and C. cadaveris. Examples of microorganisms belonging to the Tindallia genus include T. cariforniensis, T. magadiensis, and T. texcoconensis.

[0028] Microorganisms belonging to the Fusobacteriaceae family may also belong to the Fusobacterium genus. Examples of microorganisms belonging to Fusobacterium include F. varium, F. necrophorum, F. nucleatum, and F. russii.

[0029] Microorganisms belonging to the Morganellaceae family may also belong to the Morganella genus. Examples of microorganisms belonging to the Morganella genus include M. morganii and M. psychrotolerans.

[0030] Microorganisms belonging to the Pasteurellaceae family may be microorganisms belonging to either the Aggregatibacter genus or the Pasteurella genus. Examples of microorganisms belonging to the Aggregatibacter genus include A. actinomycetemcomitans, A. aphrophilus, and A. kilianii. Examples of microorganisms belonging to the Pasteurella genus include P. multocida and P. skyensis.

[0031] Tyrosine phenol lyase may also be a mutant enzyme obtained by introducing amino acid mutations into the wild-type enzyme derived from the aforementioned species, thereby conferring or enhancing the activity that catalyzes the reaction from tyrosine to phenol.

[0032] Preferably, genetically modified recombinants that enhance phenol production capacity include those having expressibly introduced 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase gene, transketolase gene, chorismic acid mutase / prephenic acid dehydrogenase gene, phosphoenolpyruvate synthase gene, and tyrosine phenol lyase gene. Furthermore, mutant strains having a gene mutation introduced into the parent strain, using this recombinant strain as the parent strain, which show improved growth compared to the parent strain when cultured in a phenol-containing culture medium and exhibit enhanced phenol production capacity when used in biomass fermentation, can also be preferably used as genetically modified recombinants that enhance phenol production capacity. Such mutant strains can be obtained by introducing a gene mutation into the parent strain and selecting a mutant strain that shows improved growth compared to the parent strain when cultured in the presence of phenol. The introduction of gene mutations into parental plants can be carried out using conventionally known methods, such as mutation treatment using UV mutagens or mutation treatment using chemical mutagens such as ethyl methanesulfonic acid (EMS).

[0033] The culture medium is a liquid medium containing sufficient nutrients, including at least one carbon source, on which microorganisms can grow. The culture medium may also contain biomass. Biomass refers to organic resources derived from plants and animals that can be recycled into energy or matter, and excludes fossil resources. In this disclosure, biomass particularly refers to resources containing fermentable carbohydrates that can be used for microbial fermentation. Examples of fermentable carbohydrates, though not particularly limited, include glucose, xylose, sucrose, starch, molasses, glycerol, ribitol, and erythritol. The biomass may also include lipids, amino acids, organic acids, and alcohols that can be derived from or produced by the metabolism of these fermentable carbohydrates. These biomass can be used individually or in combination of two or more. The concentration of biomass in the culture medium is not particularly limited as long as it can produce phenols. The concentration is, for example, 0.05 to 20 (w / v)%, preferably 0.1 to 15 (w / v)%, and more preferably 0.2 to 10 (w / v)%. Using 0.2 (w / v)% or more increases the tyrosine production capacity of microorganisms, while using 10 (w / v)% or less is because adding more than that does not result in a dramatic increase in effect, or inhibits the growth and metabolism of microorganisms. Furthermore, during cultivation, additional biomass may be added in accordance with the decrease in biomass.

[0034] Microbial culture is carried out in a mixture of culture medium and organic solvent, and stirring, temperature conditions, culture time, etc., can be set as appropriate based on conventionally known methods. Culture may be a process that involves microbial growth or a process that does not involve growth (resting cell reaction), but the former is more preferable. There are no particular restrictions on the timing of organic solvent addition, but in order to achieve both cell growth and phenol production, it is preferable to add it when phenol production starts rather than at the start of culture. For example, when controlling the expression of tyrosine phenol lyase placed under the control of the lac promoter using isopropyl β-D-1-thiogalactopyranoside (IPTG), it is preferable to add the organic solvent at the same time as adding IPTG. In the case of two-phase culture using a culture carrier containing culture medium and organic solvent, phenols produced by microorganisms migrate into and accumulate in the organic solvent.

[0035] The organic solvent is preferably one in which the Hansen solubility parameter (HSP) distance with water is 38.0 to 45.0 and the HSP distance with phenol is 5.0 to 14.0. It is believed that such an organic solvent can effectively transfer phenols produced by microorganisms into the organic solvent while suppressing inhibition of microbial growth. If phenols produced by microorganisms do not sufficiently transfer into the organic solvent and remain in the culture medium, it will cause inhibition of microbial growth by the phenols. Therefore, by effectively transferring phenols into the organic solvent, it is expected that microbial growth and phenol production will be promoted, increasing the amount of phenols recovered from the organic phase in step 3 and improving the productivity of phenols. The HSP distance of the organic solvent to water is preferably 39.0 to 45.0, more preferably 40.0 to 44.0, and even more preferably 40.0 to 42.0. The HSP distance of the organic solvent to phenol is preferably 8.0 to 14.0, more preferably 10.0 to 14.0, and even more preferably 11.0 to 13.0. The HSP distance to the organic solvent is preferably 39.0 to 42.0 with respect to water and 11.0 to 14.0 with respect to phenol, or 39.0 to 42.0 with respect to water and 11.0 to 13.0 with respect to phenol.

[0036] Here, the Hansen solubility parameter (HSP) is one of the indicators used to determine the solubility of multiple compounds, that is, the strength of the affinity between multiple compounds. For example, if the HSP distance between two compounds is small, the solubility of those compounds tends to be high. The HSP of a mixture of multiple compounds can be determined by summing the products of the HSP of each compound in the mixture and their composition ratios. The definition and calculation method of the Hansen solubility parameter are described in Charles M. Hansen's "Hansen Solubility Parameters: A User's Handbook" (CRC Press, 2007).

[0037] The Hansen solubility parameter is divided into a dispersion force term (δD) corresponding to van der Waals interactions, a polarity term (δP) due to attractive and repulsive forces arising from the dipole moment, and a hydrogen bonding term (δH) due to hydrogen bonding generated by active hydrogen and lone pairs of electrons. The Hansen solubility parameter distance (dHSP) of two compounds a and b can be calculated using the following formula (1) based on the components of their respective solubility parameters. In formula (1), the units of δD, δP, and δH are all (MPa). 1 / 2 (dHSP) 2 =4×(δDa−δDb) 2 + (δPa - δPb) 2 + (δHa - δHb) 2 …(1)

[0038] In this specification, the Hansen solubility parameter distances (dHSP) between water and phenol and each organic solvent were calculated using the computer software Hansen Solubility Parameters in Practice 5th Edition 5.4.08 (HSPiP 5th Edition 5.4.08).

[0039] Examples of organic solvents possessing the above-mentioned HSP distance include compounds that can be used as plasticizers, particularly plasticizers for vinyl chloride resins, and preferably ester compounds that can be used as plasticizers for vinyl chloride resins. More specifically, at least one selected from the group consisting of trialkyl phosphates, diadipate diesters, dibutyrate diesters, dibacate diesters, diacetate diesters, phthalate esters, terephthalate esters, and trimetate esters can be used as the organic solvent. Among these, examples of trialkyl phosphates include tributyl phosphate, tris(2-ethylhexyl) phosphate, triphenyl phosphate, 2-ethylhexyldiphenyl phosphate, trityl phosphate, triamyl phosphate, tri-o-cresyl phosphate, cresyldiphenyl phosphate, etc., and preferably tributyl phosphate and tris(2-ethylhexyl) phosphate, and particularly preferably tris(2-ethylhexyl) phosphate. Examples of adipate diesters include diisononyl adipate, di(2-ethylhexyl) adipate, dioctyl adipate, diisodecyl adipate, and diisobutyl adipate, with diisononyl adipate, diisobutyl adipate, bis(2-ethylhexyl) adipate, and diisodecyl adipate being preferred. Examples of butyrate diesters include 2,2,4-trimethylpentane-1,3-diyldiisobutyrate and ethylene glycol dibutyrate, with 2,2,4-trimethylpentane-1,3-diyldiisobutyrate being preferred. Dibutyl sebacate is preferred as a sebacate diester. 2-ethylhexyl acetate is preferred as an acetate diester. These organic solvents can also be used as extractants for phenols from culture solutions of microorganisms capable of producing phenols, and can be used individually or in combination of two or more. From the viewpoint of phenol productivity, tris(2-ethylhexyl) phosphate and / or tributyl phosphate are particularly preferred as organic solvents.

[0040] As the organic solvent having the above HSP distance, 2,2,4-trimethylpentane-1,3-diyl diisobutyrate (TXIB, HSP distance from water 41.8, HSP distance from phenol 14.0), tris(2-ethylhexyl) phosphate (TEP, same 40.3, 12.3), bis(2-ethylhexyl) adipate (DEHA, same 41.2, 12.9), diisodecyl adipate (DIDA, same 42.0, 13.9), and isostearyl alcohol (same 40.2, 12.2) can be mentioned. These can be used alone or in combination of two or more. From the viewpoint of the productivity of phenols, tris(2-ethylhexyl) phosphate (TEP) and / or 2,2,4-trimethylpentane-1,3-diyl diisobutyrate (TXIB) is preferable as the organic solvent, and TXIB is particularly preferable.

[0041] When the organic solvent having the above HSP distance is used, the phenol production rate when culturing phenol-producing bacteria for the first time in a mixed solution with the culture broth, and the phenol production rate (second time / first time ratio) when performing the second culture using the phenol-producing bacteria separated and recovered from the culture of the first culture can exceed, for example, 75%, and can be 80% or more, 85% or more, 90% or more, 95% or more, or 100% or more. The second time / first time ratio when using TXIB can be, for example, 90% or more, 95% or more, or 100% or more. The second time / first time ratio when using TEP can exceed, for example, 75%, and can be 80% or more or 85% or more.

[0042] On the other hand, the second time / first time ratio when using an organic solvent not having the above HSP distance can be, for example, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less. The second time / first time ratio when using tributyrin (HSP distance from water 37.8, HSP distance from phenol 9.7) can be, for example, 75% or less, 70% or less, or 65% or less.

[0043] In particular, as the organic solvent for two-phase culture, compounds that can be used as plasticizers for vinyl chloride resins are preferably used, and ester compounds that can be used as plasticizers for vinyl chloride resins are especially preferably used. With such organic solvents, it is thought that phenols produced by microorganisms can be effectively transferred into the organic solvent while suppressing inhibition of microbial growth. If phenols produced by microorganisms do not sufficiently transfer into the organic solvent and remain in the culture medium, it will cause inhibition of microbial growth by the phenols. Therefore, by effectively transferring phenols into the organic solvent, it is expected that microbial growth will be promoted, the amount of phenols recovered from the organic solvent in step 2 will increase, and the productivity of phenols can be improved.

[0044] More specifically, as the organic solvent, at least one selected from the group consisting of trialkyl phosphates, adipic acid diesters, butyric acid diesters, phthalic acid esters, terephthalic acid esters, and trimellitic acid esters can be used. Among these, examples of the trialkyl phosphate include tributyl phosphate, tris(2-ethylhexyl) phosphate, triphenyl phosphate, 2-ethylhexyl diphenyl phosphate, tritolyl phosphate, triamyl phosphate, tri-o-cresyl phosphate, cresyl diphenyl phosphate, etc., preferably tributyl phosphate and tris(2-ethylhexyl) phosphate, and particularly preferably tris(2-ethylhexyl) phosphate. Examples of the adipic acid diester include diisononyl adipate, di(2-ethylhexyl) adipate, dioctyl adipate, diisodecyl adipate, etc., and preferably diisononyl adipate is used. Examples of the butyric acid diester include 2,2,4-trimethylpentane-1,3-diyl diisobutyrate, ethylene glycol dibutyrate, etc., and preferably 2,2,4-trimethylpentane-1,3-diyl diisobutyrate is used. These organic solvents can also be used as extractants for phenols from the cultures of microorganisms having the ability to produce phenols, and one kind can be used alone or two or more kinds can be used in combination. From the viewpoint of the productivity of phenols, particularly tris(2-ethylhexyl) phosphate, tributyl phosphate, and / or 2,2,4-trimethylpentane-1,3-diyl diisobutyrate are preferable.

[0045] In order to promote the transfer of phenols into the organic solvent and suppress the growth inhibition of microorganisms, the ratio of the organic solvent in the mixed solution is preferably 90% or less, more preferably 70% or less, and even more preferably 50% or less. In addition, since triacylglycerol containing tributyrin, etc. is assimilated (decomposed) by the lipase possessed by microorganisms, it is not preferable to use it in the two-phase culture of the present invention.

[0046] Step 2 In this step, from the culture obtained in Step 1, (1) first the bacterial cells are separated, and then the aqueous phase and organic phase are separated; (2) first the organic phase is separated, and then the aqueous phase and bacterial cells are separated; or (3) the organic phase, aqueous phase and bacterial cells are separated in a single procedure. The separation procedure may also be (4) after the aqueous phase is separated from the culture obtained in Step 1, the organic phase and bacterial cells may be separated. The separation of the aqueous phase, organic phase and bacterial cells from the culture can be carried out by appropriately combining well-known operations such as centrifugation and membrane separation as needed, and is preferably carried out by centrifugation.

[0047] The isolation of bacterial cells from the culture can also be performed by filtration.

[0048] The isolated bacterial cells can be subjected to the culture process in step 1 again and reused cyclically in steps 1 and 2.

[0049] The separated culture medium can be reused in the culture in step 1 and can be cyclically used in steps 1 and 2. In this case, the culture medium (aqueous phase) may be treated with activated carbon and / or ion exchange resin, or it may be used in the culture in step 1 without treatment. Activated carbon treatment and / or ion exchange resin treatment can remove microorganisms and impurities such as proteins, peptides, organic acids, amino acids, lipids, nucleic acids, polysaccharides, and metal ions derived therefrom. When the culture medium is reused without treatment, it may be used with some or all of the microorganisms remaining in the culture medium. When the culture medium is reused, it may be used as part of the culture medium used in step 1, or as the whole of it. In addition, various nutrients may be added to the reused culture medium.

[0050] Step 3: In this step, phenols are recovered from the organic phase obtained in Step 2. The recovery of phenols from the organic phase can be carried out by appropriately combining well-known operations such as filtration, centrifugation, vacuum concentration, ion exchange or adsorption chromatography, solvent extraction, distillation, and crystallization as needed, and is preferably carried out by distillation.

[0051] The organic solvent after phenol recovery can be reused as the organic solvent in the culture in step 1 and can be cyclically used in steps 1 and 2. When reusing the organic solvent, it may be used as part of the organic solvent used in step 1, or it may be used all of it. By reusing the culture medium, or by reusing the organic solvent in addition to the culture medium, it becomes possible to produce phenols at a lower cost.

[0052] When recovering phenols by distillation, the amount of phenols obtained can be improved by washing off phenols adhering to the equipment (condensing tubes, V-tubes, T-tubes, connecting tubes, etc.) with an organic solvent.

[0053] The recovered phenols can be detected using conventional methods such as high-performance liquid chromatography and LC / MS. When biomass is used as the starting material, the resulting phenols contain carbon-14 radioactive isotopes derived from the biomass. The carbon-14 radioactive isotope content in the phenols is at a level equivalent to that of the biomass in the starting material. Specifically, the constituent carbon atoms are carbon-10. 12 Each molecule contains 0.8 or more, preferably 1 or more, carbon-14 radioactive isotopes. Known methods can be used to measure the carbon-14 radioactive isotope content, such as gas proportional counting, liquid scintillation counting, and accelerator mass spectrometry (AMS). Methylphenols obtained from petroleum resources as starting materials do not contain carbon-14 radioactive isotopes.

[0054] [Test Example 1] 1. Phenol-producing bacteria MCP1.1.1 strain was used as the phenol-producing bacteria. MCP1.1.1 strain is a mutant strain that has acquired high phenol resistance by a mutagenesis method using a chemical mutagen, with the phenol-high producing strain MCP1.1 as the parent strain. MCP1.1 strain is a mutant strain that has acquired high phenol resistance by a mutagenesis method using ultraviolet light, with the phenol-producing strain MCP1 as the parent strain. MCP1 strain is a genetically modified strain in which tyrosine phenol lyase (TPL) derived from Citrobacter freundii has been introduced into the tyrosine-high producing strain Tyr0. The tyrosine-high-producing strain Tyr0 is a genetically modified strain of Escherichia coli BL21(DE3) into which the colismic acid mutase / prephenic acid dehydrogenase (tyrA) mutant gene (A354V / M531L), the 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase (aroG) mutant gene (D146N), the transketolase (tktA) gene, and the phosphoenolpyruvate synthase (ppsA) gene have been introduced. The MCP1.1.1 strain is internationally deposited with the Patent Microbial Depository Center (NPMD) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture), which is a depositary institution under Articles 27-2 and 27-3 of the Patent Law Enforcement Regulations and an international depositary authority under the Budapest Convention on the International Recognition of the Deposit of Microorganisms. The accession number for the above shares is NITE BP-03815, and the original deposit date is January 31, 2023.

[0055] 2. Phenol Production Reaction A loopful of MCP1.1.1 strain was taken from a freeze stock and placed in a test tube. 25 mL of prepared M9 medium or YT medium (containing 50 mg / L kanamycin sulfate) was placed in a 200 mL baffled flask and cultured with shaking at 30°C and 180 rpm for 20 hours. The resulting culture solution was inoculated into a 1 L jar fermenter (Able) containing 225 mL of M9 medium or YT medium to a concentration of 10%, and cultivation was started under controlled conditions of 30°C, 500 rpm stirring speed, and pH 7.0. 6 to 16 hours after the start of cultivation, 0.3 mM IPTG and 250 mL of each organic solvent shown in Table 2 were added, and cultivation in the mixture was started. Glucose was initially added at 20 g / L, and during cultivation, glucose was added so that the glucose concentration did not exceed 40 g / L. Samples were taken every 24 hours, and 1 mL of the mixture was centrifuged (12,000 rpm, 1 minute) to separate it into an organic phase and an aqueous phase. The organic phase was diluted 50-fold with acetonitrile, and the aqueous phase was diluted 10-fold with Milli-Q water to prepare the analytical samples.

[0056] 3. Evaluation of Phenol Productivity The phenol concentration in the organic phase was measured using UPLC-MS (Waters). An Acquity UPLC BEH C18 1.7μm 2.1×50mm (Waters) column was used. Water containing 0.1% formic acid and acetonitrile (flow rate 0.2 mL / min, column temperature 40°C) were used as the mobile phase, and elution was performed using the gradient program shown in Table 1. Phenol was detected and quantified by monitoring UV absorption at 271 nm.

[0057]

[0058] Table 2 shows the phenol production in each culture system over a 4-hour period from 140 hours to 144 hours after the start of culture.

[0059] 4. Evaluation of the physical properties and bacterial toxicity of organic solvents. For each organic solvent shown in Table 2, the Hansen solubility parameter and bacterial toxicity were evaluated by the following method.

[0060] (1) δD, δP, δH The Hansen solubility parameters for water, phenol, and each organic solvent were calculated using the computer software Hansen Solubility Parameters in Practice 5th Edition 5.4.08 (HSPiP 5th Edition 5.4.08). For solvents included in the HSPiP 5th Edition 5.4.08 database, the HSP from the database was used, and for solvents not included, the HSP estimated using the built-in Y-MB method was used.

[0061] (2) The Hansen solubility parameter distance (dHSP) was calculated using formula (1) from the Hansen solubility parameter distance database or from the estimated HSPs of water, phenol, and each organic solvent.

[0062] (3) Cellular toxicity: MCP1.1.1 strain was pre-cultured in LB medium at 30°C for 18 hours. MCP1.1.1 strain was inoculated into a 20 mL glass vial containing 800 μL of LB medium, 200 μL of organic solvent was added, and the culture was incubated at 37°C and 160 rpm. A control sample without the addition of organic solvent was prepared.

[0063] Turbidity method (OD 600 The effects of each organic solvent on the growth of phenol-producing bacteria were investigated by measurement and evaluated according to the following evaluation criteria A to D. A: No bacterial toxicity was observed (93% or more compared to the control OD). B: Little to no bacterial toxicity was observed (80% or more to less than 93% compared to the control OD). C: Slight bacterial toxicity was observed but it did not pose a practical problem (50% or more to less than 80% compared to the control OD). D: Bacterial toxicity was observed (50% or less compared to the control OD).

[0064]

[0065] Tris(2-ethylhexyl) phosphate, tributyl phosphate, 2,2,4-trimethylpentane-1,3-diyldiisobutyrate, and diisononyl adipate, diisobutyl adipate, bis(2-ethylhexyl) adipate (DEHA), dibutyl sebacate, 2-ethylhexyl acetate, and diisodecyl adipate (DIDA) did not affect microbial growth and produced high levels of phenol. All nine of these organic solvents are compounds that can be used as plasticizers for vinyl chloride resins. On the other hand, isostearyl alcohol, which did not meet the parameters, did not affect microbial growth, but produced less phenol. Similarly, triptylin and 1-decanol, which did not meet the parameters, had little effect on microbial growth, but produced less phenol. In these organic solvents, it was presumed that the phenol produced by the microorganisms did not sufficiently migrate into the organic solvent and remained in the culture medium, resulting in inhibition of microbial growth by phenol. Hexane and cumene produced zero phenol. These organic solvents were thought to be due to their strong inhibition of phenol-producing bacteria growth and high bacterial toxicity.

[0066] [Test Example 2] 1. Phenol Production Reaction 1 A loopful of MCP1.1.1 strain was taken from a freeze stock and placed in a 14 mL culture tube containing 2 mL of LB medium or M9 medium (containing 50 mg / L kanamycin sulfate). The culture was incubated at 30°C and 200 rpm for 20 hours with shaking. The entire culture solution was transferred to a 200 mL baffled flask containing 25 mL of LB medium or M9 medium (containing 50 mg / L kanamycin sulfate) and incubated at 30°C and 180 rpm for 20 hours with shaking. The resulting culture solution was inoculated into a 1 L jar fermenter (Able) containing 225 mL of 2 × YT medium or M9 medium to a concentration of 10%. Cultivation was started under controlled conditions of a culture temperature of 30°C, a stirring speed of 500 rpm, and pH 7.0. Six hours after the start of cultivation, 0.3 mM IPTG was added, followed by the addition of 250 mL of tris(2-ethylhexyl) phosphate, and cultivation was continued. Glucose was initially added at a concentration of 20 g / L, and during culture, glucose powder was added to prevent the glucose concentration from exceeding 40 g / L. 72 hours after the start of culture, the culture medium was collected and centrifuged at 8000 g for 10 minutes at room temperature. The resulting supernatant was separated into an organic solvent (organic phase) and a culture medium (aqueous phase), and the culture medium was sterilized using a filter (0.22 μm).

[0067] 2-1. Activated Carbon Treatment of Culture Medium 250 mL of the culture medium (aqueous phase) recovered in step 2 above was placed in a 500 mL Erlenmeyer flask, and activated carbon was added at a ratio of 5 wt% relative to the culture medium. After stirring with a stirring bar for 1 hour, it was allowed to stand for 23 hours. After standing, the activated carbon was removed from the culture medium (aqueous phase) using quantitative filter paper (No. 5C), and then fine powdered activated carbon was removed using a syringe filter (PTFE, 0.45 μm).

[0068] 2-2. IER treatment of the culture medium Place 250 mL of the culture medium (aqueous phase) recovered in step 2 above into a 500 mL Erlenmeyer flask and treat with basic ion exchange resin (DIAION TM PA306 (conditioned) was added to the culture medium at a rate of 5 wt%. After stirring with a stirring bar for 1 hour, the mixture was allowed to stand for 23 hours. From the culture medium (aqueous phase) after standing, the basic ion exchange resin was removed using quantitative filter paper (No. 5C), and then the crushed basic ion exchange resin was removed using a syringe filter (PTFE, 0.45 μm).

[0069] 3-1. Phenol Production Reaction 2-1 (Fermenter Culture) Fermenter culture was performed using 50% activated carbon treated medium, IER treated medium, or untreated medium. Specifically, one loopful from the freeze stock of MCP1.1.1 strain was placed in a 14 mL culture tube containing 2 mL of LB medium or M9 medium (containing 50 mg / L kanamycin sulfate), and cultured with shaking at 30°C and 200 rpm for 20 hours. The entire culture solution was transferred to a 200 mL baffled flask containing 25 mL of LB medium or M9 medium (containing 50 mg / L kanamycin sulfate), and cultured with shaking at 30°C and 180 rpm for 20 hours. The obtained culture solution was inoculated into a 1 L jar fermenter (Able) containing 100 mL of 2×YT medium or M9 medium, and 125 mL of activated carbon-treated medium, IER-treated medium, or untreated medium. Cultivation was started under controlled conditions of a culture temperature of 30°C, a stirring speed of 500 rpm, and pH 7.0. Six hours after the start of cultivation, 0.3 mM IPTG was added, followed by the addition of 250 mL of tris(2-ethylhexyl) phosphate, and cultivation was continued. Glucose was initially added at 20 g / L, and during cultivation, glucose powder was added to prevent the glucose concentration from exceeding 40 g / L. Seventy-two hours after the start of cultivation, the culture solution was collected and centrifuged at 8000 g for 10 minutes at room temperature. The obtained supernatant was separated into organic solvent (organic phase) and medium (aqueous phase), and the medium was sterilized using a filter (0.22 μm).

[0070] Sampling was performed every 24 hours, and phenol concentration was measured. Phenol concentration was measured using UPLC-MS (Waters). An Acquity UPLC BEH C18 1.7μm 2.1×50mm (Waters) column was used. Water containing 0.1% formic acid and acetonitrile (flow rate 0.2 mL / min, column temperature 40°C) were used as the mobile phase, and elution was performed using the gradient program shown in Table 1. Phenol was detected and quantified by monitoring UV absorption at 271 nm. Phenol production was calculated using the following formula: Phenol production (g / L) = (Phenol concentration in organic phase × Volume of organic phase liquid + Phenol concentration in aqueous phase × Volume of aqueous phase liquid) / Volume of aqueous phase liquid

[0071] When the culture medium (aqueous phase) separated in step 2 was reused in 50% of the culture medium from step 1 without treatment (50% wastewater (untreated)), phenol production was found to be equivalent to or greater than that of the case without reused culture medium (CTRL) after 144 hours from the start of cultivation. Furthermore, when the culture medium (aqueous phase) separated in step 2 was treated with activated carbon and then reused in 50% of the culture medium from step 1 (50% wastewater (activated carbon treated)), phenol production was found to be 50% or more greater than that of the case without reused culture medium (CTRL) after 144 hours from the start of cultivation.

[0072] 3-2. Phenol Production Reaction 2-2 (Flask Culture) A loopful of MCP1.1.1 strain was taken from the freeze stock and placed in a 14 mL culture tube containing 2 mL of LB medium or M9 medium (containing 50 mg / L kanamycin sulfate). The culture was incubated at 30°C and 200 rpm for 20 hours with shaking. The culture was then transferred to a 200 mL baffled flask containing 10 mL of medium, each containing 50% LB medium or M9 medium (containing 50 mg / L kanamycin sulfate), 50% activated carbon treated medium, 50% IER treated medium, or 50% untreated medium. The culture was incubated at 30°C and 180 rpm with shaking. Six hours after the start of culture, 0.3 mM IPTG and 10 mL of tris(2-ethylhexyl) phosphate were added to start the culture. Glucose was initially added at 20 g / L, and during culture, glucose powder was added to prevent the glucose concentration from exceeding 40 g / L.

[0073] Sampling was performed 24 and 88 hours after the start of culture, and phenol concentration was measured.

[0074] The results are shown in Figure 1. When the culture medium (aqueous phase) separated in step 2 was reused without treatment (untreated wastewater), the amount of phenol produced after 24 hours of culture was equivalent to that of the control (water) without reused culture medium. Furthermore, after 88 hours of culture, the amount of phenol produced was more than 150% of that of the control. When the culture medium (aqueous phase) separated in step 2 was treated with activated carbon (activated carbon treated wastewater) or ion exchange resin (IER treated wastewater) and then reused, the amount of phenol produced was equivalent to that of the control after 24 hours of culture, and more than 200% of that of the control after 88 hours.

[0075] [Test Example 3] 1. First Phenol Production Reaction One loopful of MCP1.1.1 strain was taken from the freeze stock and placed in a 200 mL baffled flask containing 25 mL of M9 medium (containing 50 mg / L kanamycin sulfate). The culture was incubated at 30°C and 180 rpm for 20 hours with shaking. The resulting culture was inoculated into a 1 L jar fermenter (Able) containing 225 mL of M9 medium to a concentration of 10%, and incubation was started under controlled conditions of 30°C, 500 rpm, and pH 7.0 with shaking. Six hours after the start of incubation, 0.3 mM IPTG and 250 mL of each organic solvent shown in Table 3 were added, and two-phase incubation in the mixture was started. Glucose was initially added at 20 g / L, and during incubation, glucose powder was added so that the glucose concentration did not exceed 40 g / L.

[0076] Sampling was performed every 24 hours. 1 mL of the culture (mixture) was centrifuged (12,000 rpm, 1 minute) to separate the organic phase and aqueous phase. The organic phase was diluted 50-fold with acetonitrile, and the aqueous phase was diluted 10-fold with Milli-Q water to prepare the analytical samples. The culture was terminated approximately 250 hours after the start of cultivation.

[0077]

[0078] 2. Evaluation of Phenol Productivity Phenol concentrations in the organic and aqueous phases were measured using UPLC-MS (Waters). An Acquity UPLC BEH C18 1.7μm 2.1×50mm column (Waters) was used. Water containing 0.1% formic acid and acetonitrile (flow rate 0.2 mL / min, column temperature 40°C) were used as the mobile phase. Elution was performed using the gradient program shown in Table 4, and phenol was detected and quantified by monitoring UV absorption at 271 nm. The phenol production rate was calculated using the following formula: Phenol production rate = [(Phenol concentration in organic phase × Volume of organic phase liquid + Phenol concentration in aqueous phase × Volume of aqueous phase liquid) / Volume of aqueous phase liquid] / Culture time

[0079]

[0080] 3. Second phenol production reaction using recovered bacterial cells The culture (mixture) after the "phenol production reaction" described in 2 above was transferred to a centrifuge tube and centrifuged (3,000 g, 10 minutes) to separate and recover the aqueous phase, organic phase, and bacterial cells. The total amount of recovered bacterial cells was suspended in 250 mL of M9 medium (0.3 mM IPTG, 50 mg / L Kan) and transferred to a 1 L jar fermenter. 250 mL of the same organic solvent as in the first phenol production reaction was added, and two-phase culture in the mixture was started again. Glucose was initially added at 20 g / L, and during culture, glucose powder was added so that the glucose concentration did not exceed 40 g / L. Sampling was performed every 24 hours, and the phenol concentrations in the organic phase and aqueous phase were measured in the same manner as in "Evaluation of phenol productivity" described in 3 above.

[0081] Figure 2 shows the phenol production rates in the first and second phenol production reactions. When 2,2,4-trimethylpentane-1,3-diyldiisobutyrate (TXIB) and tris(2-ethylhexyl) phosphate (TEP) were used, the phenol production rate in the second culture exceeded 80% of that in the first culture. On the other hand, when triptylin was used, the phenol production rate in the second culture decreased to less than 80% of that in the first culture. It is presumed that the high phenol productivity was obtained with 2,2,4-trimethylpentane-1,3-diyldiisobutyrate (TXIB) and tris(2-ethylhexyl) phosphate (TEP) compared to triptylin because the inhibition of microbial growth due to exposure to organic solvents was suppressed.

Claims

1. A method for producing phenols, comprising step 1 of culturing a microorganism capable of producing phenols in a mixture of culture medium and an organic solvent, wherein the Hansen solubility parameter (HSP) distance of the organic solvent with water is 38.0 or higher, and the HSP distance with phenol is 14.0 or lower.

2. The production method according to claim 1, wherein the organic solvent is an ester compound.

3. The method for producing phenols according to claim 2, wherein the organic solvent is a compound that can be used as a plasticizer.

4. The manufacturing method according to claim 3, wherein the plasticizer is a plasticizer for vinyl chloride resins.

5. The manufacturing method according to claim 2, wherein the organic solvent is at least one selected from the group consisting of trialkyl phosphate, diadipate, dibutyrate, dibacate, and diester acetate.

6. The manufacturing method according to claim 5, wherein the organic solvent is at least one selected from the group consisting of tributyl phosphate, tris(2-ethylhexyl) phosphate, triphenyl phosphate, 2-ethylhexyldiphenyl phosphate, trityl phosphate, triamyl phosphate, tri-o-cresyl phosphate, cresyldiphenyl phosphate, diisononyl adipate, bis(2-ethylhexyl) adipate, dioctyl adipate, diisodecyl adipate, 2,2,4-trimethylpentane-1,3-diyldiisobutyrate, ethylene glycol dibutyrate, diisobutyl adipate, bis(2-ethylhexyl) adipate, dibutyl sebacate, and acetic acid (2-ethylhexyl).

7. The manufacturing method according to claim 1, wherein the phenols are at least one selected from the group consisting of phenol, catechol, cresol, xylenol, 4-hydroxybenzoic acid, and salicylic acid.

8. The manufacturing method according to claim 1, further comprising: step 2 of separating at least an organic solvent from the culture obtained in step 1; and step 3 of recovering phenols from the separated organic solvent.

9. The manufacturing method according to claim 8, further comprising the step of separating bacterial cells from the culture obtained in step 1, wherein the separated bacterial cells are subjected to the culture in step 1 again.

10. A method for producing phenols according to claim 8, comprising a step of further separating the culture medium from the culture obtained in step 1, wherein the separated culture medium is again used for culturing in step 1.

11. The manufacturing method according to claim 10, wherein the separated culture medium is subjected to treatment with activated carbon and / or ion exchange resin, or is subjected to the culture again in step 1 without treatment.

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