JP2020065494A - System for producing trihydroxybenzene - Google Patents

Abstract

To provide a system for producing THB with high efficiency.SOLUTION: Provided is a system for producing trihydroxybenzene (THB), comprising: a culture tank that holds a liquid medium containing bacteria synthesizing deoxy-scyllo-inosose (DOI) and cultures the bacteria to produce a bacterial culture; and a heating device that is connected to the culture tank and heats the bacterial culture at a high temperature of 80°C or higher to produce a product solution containing trihydroxybenzene (THB).SELECTED DRAWING: None

Description

  The present invention relates to a system for producing trihydroxybenzene (THB).

  Deoxyscylinosose (2-deoxy-scyllo-inosose, abbreviated as DOI) is a compound that can be induced into various useful chemical substances. Generally, industrially useful chemical substances, particularly carbon six-membered ring compounds, are often chemically produced from fossil resources, especially petroleum. However, since it is known that DOI can be synthesized from saccharides by a bioprocess using a bacterium that expresses a specific enzyme (Patent Documents 1 to 3), from the viewpoint of avoiding dependence on fossil resources and protecting the global environment, It is a particularly attractive chemical substance.

  That is, a plant material mainly composed of cellulose, such as wood chips, is decomposed into a monosaccharide such as glucose by a known technique, and the monosaccharide is converted into a DOI by the bioprocess described above. It is possible to realize chemical product manufacturing that effectively uses fossil resources.

  Of the additional intermediates that can be derived from DOI, one of the most representative is trihydroxybenzene (THB). THB is a compound produced by a dehydration reaction from DOI (a total of two molecules of water are eliminated from one molecule of DOI). THB can be further derivatized into useful compounds such as quercitol, carbaglucose, catechol, adipic acid derivatives, hydroquinone, trimethoxybenzene, trimethoxybenzoic acid phenyl ester and sesamol. These THB-derived useful compounds can be used for applications such as antidiabetic agents, nylons, photographic agents, polymerization inhibitors, Li secondary battery additives, cellulose film additives, and antidepressants (Non-Patent Document 1). ).

International Publication No. 2006/109479 International Publication No. 2010/053052 International Publication No. 2015/005451

Biotechnology, Volume 93, 533-535, 2015

  In order to obtain THB from DOI synthesized by a bioprocess using bacteria, conventionally, acidification of culture medium, centrifugation, filtration, cation exchange, anion exchange, drying, concentration, azeotropic distillation with methanol, and drying It is said that the purified powder DOI is obtained through a number of steps including crystallization, crystallization, and filtration drying, and is dissolved in water, and finally the product THB is obtained by heating at 170 ° C. for 2 hours. It was However, many such processes are laborious and time consuming, and significantly increase the manufacturing cost. It is desired to improve the efficiency of THB production by simplifying the production process and / or improving the yield.

  The present invention provides a system for producing THB with high efficiency.

  The present inventor has discovered that THB is produced in high yield by directly heating a culture solution of a bacterium that synthesizes DOI. The present invention is based on this finding.

The present invention includes at least the following embodiments.
[1]
A culture tank for holding a liquid medium containing bacteria that synthesize deoxyscylinosose (DOI) and culturing the bacteria to produce a bacterial culture solution;
A heating device connected to the culture tank to heat the bacterial culture at a high temperature of 80 ° C. or higher to generate a product solution containing trihydroxybenzene (THB);
System for producing trihydroxybenzene (THB).
[2]
The culture tank is connected to the heating device via a liquid supply pipe, and the heating device heats the bacterial culture solution sent from the culture tank inside the heating device, [1] The system described in.
[3]
The system according to [2], wherein the culture tank is connected to the heating device via a bacterial cell removing device that removes bacterial cells of the bacteria from the bacterial culture solution.
[4]
The system according to [1], wherein the heating device is an integrated heating device that heats the bacterial culture solution inside the culture tank.
[5]
The system according to any one of [1] to [4], further including a purification device that removes solid substances and insoluble substances from the product solution.
[6]
The system according to any one of [1] to [5], further including a separation device that separates trihydroxybenzene (THB) from the product solution.
[7]
The system according to [6], wherein the separator comprises a solvent extractor.
[8]
The culture tank is one of a stirring device for stirring the medium, a shaking device for shaking the medium, an aeration device for aerating the medium, and a heating device for heating the medium. The system according to any one of [1] to [7], which has the above.

  The present invention significantly improves the efficiency of producing THB. That is, the number of steps of the process from the synthesis of DOI by the bacterium to the production of THB can be significantly simplified and a part or all of the process can be continuously performed.

FIG. 1 shows an outline of a THB manufacturing system according to one embodiment. FIG. 2 shows an outline of a THB manufacturing system according to another embodiment. FIG. 3 shows the appearance (a) and the DOI content (b) of the DOI fermentation liquor (stock solution) and the heated one. FIG. 4 shows a ¹³ C-NMR spectrum of a THB product solution (bottom) obtained from a DOI fermentation broth and a THB sample (top) after solvent extraction.

  In one aspect, the present disclosure includes the step of heating a bacterial culture containing deoxyscylinosose (DOI) at an elevated temperature of 80 ° C. or higher to obtain a product solution containing trihydroxybenzene (THB). A method for producing hydroxybenzene (THB) is provided.

The structure of deoxyscylinosose (DOI) is shown below.

The structure of trihydroxybenzene (THB) is shown below.

  Although the structure shown above corresponds to 1,2,4-trihydroxybenzene, the THB produced in this embodiment has 1,2,3 in addition to 1,2,4-trihydroxybenzene. -Trihydroxybenzene (pyrogallol) may also be included. The product solution may also include catechol.

  The concentration of DOI in the heated bacterial culture, that is, the concentration of DOI in the bacterial culture before heating is preferably 1 g / L or more, more preferably 10 g / L or more, More preferably, it is 50 g / L or more. The upper limit of the DOI concentration is not particularly limited, but is usually 100 g / L or less, for example, 90 g / L or less, or 80 g / L or less.

Bacterial culture is a conditioned liquid medium containing DOI obtained after culturing bacteria that synthesize DOI in liquid medium under DOI synthesis conditions. Liquid media suitable for culturing bacteria that synthesize DOI are known to those of ordinary skill in the art or can be prepared by those of ordinary skill in the art, and typically 10 in stationary phase. ^It can support culturing at a cell density of ⁷ cells / mL or more, preferably 10 ⁸ cells / mL or more. As known to those skilled in the art, a liquid medium for culturing bacteria usually contains a carbon source (in particular, sugar), a nitrogen source (such as amino acid), vitamins, inorganic salts and the like. In the present embodiment, it was confirmed that similar results can be obtained even if the specific material components are different, as long as it is a liquid medium capable of supporting the culture at a relatively high cell density and DOI synthesis as described above. ing. Although sugars other than glucose can also be metabolized to glucose, it is preferable that the liquid medium contains glucose itself because it provides a substrate for DOI synthesis directly and is efficient.

An example of a typical liquid medium that can be used to obtain the bacterial culture of this embodiment is YE medium, which contains BD Bacto Tryptone (pancreatic digest of casein) 50 per 6.5 L of water. ˜200 g (104 g in 2 × YE medium), 60 to 260 g of BD Bacto Yeast Extract (water-soluble extract of yeast) (130 g in 2 × YE medium), and 15 to 60 g of NaCl (32.5 g in 2 × YE medium). ), Glucose 200-800g (377M-390g in 2xYE medium), mannitol 150-650g (335g in 2xYE medium), and optionally antibiotics such as ampicillin, phytic acid, antifoaming agent, etc. Additives may be included in small amounts (eg, 5 g or less total). Phytic acid is an additive component capable of improving DOI production by bacteria, and for example, 50% (w / w) phytic acid aqueous solution may be added in an amount of 1 to 20 g per the above amount of YE medium.
Another specific example of the liquid medium (CSL medium) is CSL (corn steep liquor) 150 to 700 g (standard 325 g), glucose 1000 to 5000 g (standard 2010 g), fructose 1000 to 1000 per 52.6 kg of water. 5000 g (standard 2010 g), xylose 100-500 g (standard 200 g), ammonium sulfide 50-500 g (standard 170 g), ammonium chloride 20-200 g (standard 78.2 g), magnesium sulfate heptahydrate 20-200 g (standard 78.2 g), dipotassium hydrogen phosphate 40-200 g (standard 91 g), potassium dihydrogen phosphate 40-200 g (standard 91 g), iron (II) sulfate heptahydrate 2 to 10 g (4.55 g as standard) of the product. The CSL medium may further include 50 to 50 g (w / w) phytic acid in an amount of 40 to 200 g (91 g by standard) per 52.6 kg of water. The CSL medium may contain an antifoaming agent (eg 5 to 30 g of adecanol antifoaming agent per the above amount of CSL medium).
During the culture, part or all of the medium components may be supplemented at any time. For example, it is preferable to supplement the sugar component during the culture.

  The heated bacterial culture may or may not contain bacterial cells. That is, in one embodiment, the heated bacterial culture is a liquid medium after culturing the bacteria, which contains the bacteria or after the bacteria have been removed. In the present specification, the term “solution” (including “aqueous solution”) means a liquid in which at least one substance different from the solvent is dissolved or suspended. An insoluble substance or a solid substance may be suspended in the solution. The insoluble substance and the solid substance as used herein may include biological cells.

  That is, the bacterial culture solution to be heated, that is, the bacterial culture solution at the stage before being heated may contain bacterial cells of DOI-synthesized bacteria. For example, after culturing bacteria in a liquid medium to synthesize DOI from sugar, the liquid medium containing the bacteria and the DOI (that is, the bacterial culture solution) is kept at high temperature as it is without removing the bacteria and purifying the DOI. The method of the present embodiment can be carried out by heating. Such an embodiment allows THB to be produced and used bacteria inactivated / sterilized at the same time, which not only greatly simplifies the overall procedure, but also in terms of biosafety and hygiene. Will also be advantageous.

  In one embodiment, the method further comprises culturing the DOI-synthesizing bacteria in the liquid medium under DOI synthesis conditions, prior to the step of heating the bacterial culture. The liquid medium after culturing provides the bacterial broth to be heated.

The bacterial broth to be heated may be one that has been cultured for preferably 12 hours or more, more preferably 16 hours or more, for example, 24 hours or more, or 32 hours or more. In culture, a cell density of preferably 10 ⁷ cells / mL or more, more preferably 10 ⁸ cells / mL or more can be achieved within 24 hours from a cell density of 10 ⁴ cells / mL or less. By culturing a bacterium that synthesizes DOI, preferably 1 g / L or more, more preferably 10 g / L or more, still more preferably 50 g / L or more DOI accumulates in the bacterial culture. The culture temperature is usually 25 to 38 ° C, preferably 30 to 37 ° C. Culture conditions for synthesizing DOI in bacteria can be appropriately adjusted by those skilled in the art based on ordinary knowledge.

  Bacteria that synthesize DOI are known to those skilled in the art, or can be identified or obtained by those skilled in the art based on known information. Specific examples of such bacterial species include E. coli, Bacillus circulans, Bacillus amyloliquefaciens, Bacillus subtilis, Corynebacterium glutamicum, and Geobacillus stearothermophilus. Not limited to. E. coli is particularly preferred. The bacterium preferably expresses 2-deoxyscylinosose synthase. Most preferably, the bacterium that synthesizes DOI is E. coli that expresses 2-deoxyscylinosose synthase. A preferred example of 2-deoxyscylinosose synthase is a product of the btrC gene derived from Bacillus circulans (Patent Document 1), but as long as DOI synthetic activity is maintained, a homologous gene product derived from another bacterium. It will be understood by those skilled in the art that a modified natural sequence can be substituted. Such a gene may be endogenous, but is preferably exogenously introduced by a genetic engineering technique. Means for increasing and / or optimizing gene expression, such as plasmid vector selection, promoter selection, codon optimization, etc., are known to those of skill in the art. For example, the promoter may be a constitutively expressed promoter or an expression-inducible promoter.

  Other genes may be modified, added, or deleted in the host bacterium into which 2-deoxyscylinosose synthase is introduced. For example, one or more or all of pgi, zwf, and pgm, which are genes that metabolize glucose-6-phosphate as a substrate for DOI synthesis into another compound, and / or a protein in stationary phase The yield of DOI can be increased by using, as a host, Escherichia coli in which rmf, which is a gene involved in synthesis suppression, is destroyed (Patent Document 1). In that case, it may be necessary to add a carbon source other than glucose, such as mannitol, to the medium. However, for example, by adding a gene for sucrose hydrolase (cscA) to Escherichia coli, DOI synthesis from glucose can be efficiently performed while using inexpensive sucrose as a nutrient source (Patent Document 2). . It has been shown that these systems can accumulate at least several tens of grams of DOI per liter of medium (Patent Documents 1 to 3).

  In the liquid medium (bacterial culture solution) after the bacteria have synthesized DOI, 2-deoxyscylinosose (DOI) and one or more related compounds selected from the following are contained in small amounts (for example, in total). , 20% or less, 15% or less, 10% or less, or 5% or less by weight of 2-deoxycylinosose (DOI)): 1-epi-DOI, α, β-unsaturated ketone, (+ ) Vibo-quercitol, and scyllo-quercitol.

  The bacterial culture may be heated while the bacteria are still contained, but a step of removing bacterial cells of the bacteria from the bacterial culture may be added before heating. By this step, it is possible to avoid the possibility that a large amount of insoluble substance released from the heated microbial cell may reduce the purity of the product solution or inhibit the reaction. The technique used in the step of removing bacterial cells from the bacterial culture can be appropriately selected from those known by those skilled in the art, and examples thereof include filter filtration and centrifugation. Filter filtration is preferable because it can be performed by a relatively simple mechanism.

  In order to remove the bacterial cells, a filter having a cutoff of 1 μm or less, for example, a filter having a pore size of 0.2 μm can be used. A plurality of filtration filters having different pore sizes may be combined. By selecting the pore size of the filter, it is possible to remove other than the bacterial cells from the bacterial culture solution before heating. For example, THB purity in the product solution after heating can be improved by removing components unnecessary for THB synthesis from the bacterial broth by appropriate filtration. Unnecessary components for THB synthesis can also be removed by salting out.

  The “hot” temperature for heating the bacterial culture is 80 ° C. or higher. If the temperature is lower than 80 ° C., the DOI conversion reaction itself may occur, but it is considered that sufficient conversion efficiency cannot be obtained. The high temperature is preferably 90 ° C or higher, more preferably 100 ° C or higher, more preferably 130 ° C or higher, further preferably 140 ° C or higher, particularly preferably 160 ° C or higher, most preferably 170 ° C or higher.

  The upper limit of the high temperature is not particularly limited, but is usually 300 ° C. or lower, or 200 ° C. or lower. The elevated temperature is typically 130-180 ° C. High temperatures may be combined with high pressures above atmospheric pressure. Such high pressure is, for example, 1 MPa or less, 0.8 MPa or less, 0.5 MPa or less, or 0.2 MPa or less.

  The heating time of the bacterial culture at elevated temperature may vary depending on the temperature used and the desired THB yield, but is usually at least 30 seconds, preferably at least 1 minute, more preferably at least 2 minutes, more preferably at least 5 minutes. , More preferably at least 10 minutes, even more preferably at least 30 minutes. The heating may be performed for 1 hour or more, 2 hours or more, or 4 hours or more. The heating may be continuous over the above time, or the cumulative time of reaching a high temperature by intermittent heating may be the above time. The upper limit of the heating time is not particularly limited, but is, for example, 24 hours or less, 4 hours or less, 2 hours or less, 1 hour or less, 30 minutes or less, or 10 minutes or less.

  Heating the bacterial culture at an elevated temperature can be accomplished using techniques known to those skilled in the art, such as injecting hot air (steam) or hot liquid into the bacterial culture, solidifying the bacterial culture. It can be achieved by contacting with a heat source, heating the inner wall of the vessel or tube containing the bacterial culture, energizing the bacterial culture and / or applying microwaves to the bacterial culture.

The atmosphere during heating of the bacterial culture is preferably air, an inert gas such as nitrogen and argon, or a mixture thereof. The atmosphere during the heating of the bacterial culture is preferably a substantially hydrogen (H ₂ ) -free atmosphere. The substantially hydrogen-free atmosphere has, for example, a hydrogen volume concentration of less than 1 ppm. The heating of the bacterial culture is preferably carried out in the substantial absence of a metal catalyst or reduction catalyst such as palladium, rhodium, ruthenium, platinum, iridium, nickel, cobalt and copper.

  "Product solution" means a solution in which THB is dissolved or suspended, which is obtained after heating the bacterial culture.

  Filtering or centrifugation may be performed to remove impurities from the product solution. In particular, when a bacterial culture solution containing bacterial cells is heated, a large amount of insoluble matter derived from the modified bacterial cells may be contained in the product solution, and thus these treatments may be particularly significant.

  The method of this embodiment may further include a step of separating THB from the product solution. A known method can be appropriately used for the THB separation. Preferably THB is extracted from the product solution by solvent extraction. The solvent extraction may be repeated once or more. As the extraction solvent, ethyl acetate or methyl acetate is preferable, and ethyl acetate is particularly preferable, but not limited thereto. The volume of the extraction solvent used for extraction is preferably equal to or larger than the volume of the product solution. Solvent extraction can be carried out by mixing the product solution with an organic solvent and then separating into an organic layer and an aqueous layer and recovering an organic layer containing THB, as will be understood by those skilled in the art. Water-soluble salts may be added to the product solution for solvent extraction. For example, adding less than saturated concentration of sodium chloride to the product solution followed by solvent extraction may improve yield. Also, the yield may be improved by adding an acid, such as hydrochloric acid, to the product solution to lower the pH before solvent extraction. The pH is preferably less than 2, more preferably less than 1.5, even more preferably less than 1. When the THB sample after separation contains insoluble matter, the insoluble matter can be removed by, for example, filter filtration.

  The THB sample after separation has a THB content of preferably 50% or more, more preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, in particular, by dry weight after removing insoluble matter and solvent. It is preferably 90% or more. The THB sample after separation has a catechol content of preferably 5% or less, more preferably 3% or less, still more preferably 2% or less, and particularly preferably 1.5% by dry weight after removing the insoluble matter and the solvent. % Or less.

  The steps from (a) the synthesis of DOI by culturing the bacteria to the production of THB by heating at high temperature and / or (b) the production of THB to the isolation of THB are carried out continuously. obtain. These steps may be performed batch by batch.

  In another aspect, the present disclosure provides a culture tank that holds a liquid medium containing DOI-synthesizing bacteria and cultures the bacteria to produce a bacterial culture, and the bacterial culture connected to the culture tank. Is heated at a high temperature of 80 ° C. or higher to produce a product solution containing trihydroxybenzene (THB), and a heating device is provided to provide a system for producing trihydroxybenzene (THB).

This system is suitable for performing the method according to the aforementioned aspects of the present disclosure. That is, what is described herein for method embodiments may be applied to system embodiments and vice versa. For example, the liquid medium is capable of supporting culture at a cell density of 10 ⁷ cells / mL or more, preferably 10 ⁸ cells / mL or more, typically in the stationary phase as described above. The bacteria are also as described above.

  The culture tank may be equipped with auxiliary equipment for providing conditions suitable for culturing bacteria. For example, the culture tank is one of a stirring device for stirring the culture medium, a shaking device for shaking the culture medium, an aeration device for aerating the culture medium, and a heating device for heating the culture medium. It may have more than one. A liquid medium supply line may be connected to the culture tank to supply a part or all of the components of the fresh liquid medium into the culture tank. In one embodiment, the liquid culture medium supply line has a switching valve so that the culture tank can be switched and connected between the liquid culture medium supply line and a liquid feeding pipe described later.

  The culture tank can be connected to the heating device via a liquid supply tube. The heating device in this case heats the liquid sent from the culture tank inside the heating device. For example, the heating device may have a structure in which a continuous heating passage is provided inside and the bacterial culture solution passing through the heating passage is heated. The heating passage can heat the liquid culture medium, for example, by injecting hot air or a hot liquid into the passing bacterial culture, or by heating the inner wall of the passage or a solid heat source arranged in the passage, The heating method is not limited to these. Hot air is, for example, heated air, steam, or nitrogen. The hot liquid is, for example, heated water or fresh liquid medium. The heating temperature, holding time, and flow rate in the heating passage can be adjusted arbitrarily. The heating passage may have a plurality of heating zones that can be set at different temperatures. The heating device may be provided with an outlet for collecting the product solution that is heated to produce THB.

  The culture tank may be connected to the heating device via a bacterial cell removing device that removes bacterial cells of the bacteria from the bacterial culture solution. The microbial cell removal device may include a filtration filter or a centrifuge. A bacterial cell removal device including a filtration filter for removing bacterial cells is preferable. A plurality of filtration filters having different pore sizes may be provided in the bacterial cell removing device, and unnecessary components other than bacterial cells may be removed at this stage. The bacterial cell removing device may be provided in the middle of the liquid feeding pipe.

  The liquid transfer tube includes one or more of a bacterial cell removal device, a pump for transferring the liquid, a safety valve for preventing excessive internal pressure (for example, 10 bar or more), and a flow rate control device for controlling the flow rate. Can have. Those skilled in the art can appropriately determine the type and number of these auxiliary equipments and the arrangement order in the direction of liquid flow.

  Alternatively, the heating device may be an integrated heating device that heats the bacterial culture solution inside the culture tank itself. The heating in this case can be achieved, for example, by injecting hot air or a hot liquid into the inside of the culture tank, or by heating the inner wall of the culture tank or a solid heat source arranged in the culture tank, but possible heating is possible. The method is not limited to these. Hot air is, for example, heated air, steam, or nitrogen. The hot liquid is, for example, heated water or fresh liquid medium.

  The heating temperature and the heating time by the heating device are as described in the embodiment of the THB manufacturing method.

  The heating device (including the culture tank provided with the heating device in the case of an integrated heating device) may be connected to a purifying device for removing solid substances and insoluble substances from the product solution. The purification device can have, for example, a filtration filter or a centrifuge. If the heated bacterial culture contains bacterial cells, the product solution may contain a large amount of insoluble matter derived from denatured bacterial cells. You can The purification device may be provided with a plurality of filtration filters having different pore sizes. Similarly, the heating device may be connected to a cooling device for reducing the temperature of the liquid. The liquid after heating at a high temperature is cooled to, for example, room temperature, and the cooling device can accelerate this cooling.

  Further, the heating device (including the fermentor in which it is provided in the case of an integrated heating device) may be connected to a separation device that separates the THB product from the product solution. The separation device is preferably connected downstream of the purification device described above. Separation devices may include known solvent extraction devices such as centrifugal extractors, mixer settlers, microreactors, flow reactors, pulse columns and the like. The solvent extraction device may have an organic layer outlet for discharging an organic layer containing THB. The solvent extraction device may further have a water layer outlet for discharging the water layer.

  The heating device may be connected to the separation device via a liquid supply pipe. This liquid supply pipe may also have an auxiliary equipment such as a pump, a safety valve, a cooling device, a purifying device, and a flow rate adjusting device as described above in any number and arrangement order.

  The entire system of the present embodiment may be integrated as one device.

  In a further aspect, the present disclosure provides a method of manufacturing THB using the THB manufacturing system of this embodiment.

  FIG. 1 shows an example of a system according to this embodiment. In the system 100 of this example, the bacteria 101 that synthesize DOI and the liquid medium 102 are held in the culture tank 103, and the culture is performed in the culture tank 103. That is, the liquid medium 102 becomes a bacterial culture liquid 102 '. The culture tank 103 can be connected to either the liquid medium supply line 105 or the liquid feeding pipe 106 by a switching valve 104. The liquid feed pipe 106 is a bacterial cell removal device 107 having a filtration filter, a pump 108 for transferring the liquid (ie, the filtered bacterial culture liquid 102 '), and an excessive internal pressure (for example, 10 bar or more) for preventing it. A safety valve 109 and a flow rate adjusting device 110 for adjusting the flow rate are provided and are connected to the heating device 111. Inside the heating device 111, the bacterial culture solution passes through the heating passage 112 and is heated at that time. In this example, three heating zones 113A, 113B, 113C are provided in the heating device 111, each having one or more heating passages so that the liquid culture medium can be heated at different temperatures. The bacterial culture solution that has been heated at a high temperature becomes a product solution containing THB, and exits the heating device 111 from the outlet 114. The outlet 114 is connected to a separation device 117 including a centrifugal extractor via a cooling device 115 for cooling the product solution and a purification device 116 having a filtration filter for removing solid substances and insoluble substances from the product solution. ing. The separation device 117 performs extraction by adding an organic solvent (eg, ethyl acetate) to the product solution and discharges the organic layer 118 and the aqueous layer 119. THB is separated in the organic layer 118.

  FIG. 2 shows another example of the system according to the present embodiment. Also in the system 200 of this example, the bacteria 201 that synthesize DOI and the liquid medium 202 are held in the culture tank 203, and the culture is performed in the culture tank 203. That is, the liquid medium 202 becomes a bacterial culture fluid 202 '. The culture tank 203 is equipped with an integrated heating device 211. The integrated heating device 211 heats the liquid culture medium 202 after culture, that is, the bacterial culture liquid 202 ′ to a high temperature by injecting hot air 220 into the culture tank 203. Therefore, the bacterial culture solution 202 ′ is converted into a THB-containing product solution in the culture tank 203. The culture tank 203 can be connected to either the liquid medium supply line 205 or the liquid feeding pipe 206 by a switching valve 204. The liquid supply pipe 206 has a pump 208 for transferring the product solution, a safety valve 209 for preventing an excessive internal pressure, a cooling device 215 for cooling the product solution, and a filtration filter for removing solid substances and insoluble substances from the product solution. It has a purifying device 216 for removal and a flow rate adjusting device 210 for adjusting the flow rate, and is connected to a separating device 217 including a centrifugal extractor. The separator 217 adds an organic solvent (for example, ethyl acetate) to the product solution to perform extraction, and discharges the organic layer 218 and the aqueous layer 219. THB is separated in the organic layer 218.

  After culturing Escherichia coli expressing 2-deoxyscylinosose synthase (btrC gene product) in 2 × YE medium under DOI synthesis conditions, the bacterial culture (hereinafter referred to as DOI fermentation broth) is kept at about 80 ° C. During the activation treatment, discoloration of the DOI fermentation broth was observed, and the present inventors speculated that the conversion reaction from DOI to THB may have occurred in this DOI fermentation broth. FIG. 3 shows DOI fermentation liquor (stock solution) at pH 6 or pH 3 and what was heated at 95 ° C. for 30 minutes, at 105 ° C. for 30 minutes, at 132 ° C. for 20 minutes, and at 132 ° C. The external appearance (a) and DOI content (b) of what was heated for 1 minute are shown. A decrease in the DOI content was observed with the discoloration of the DOI fermentation liquor, suggesting the production of THB. When these heated samples were analyzed by liquid chromatography (column: YMC-Pac ODS-AQ, eluent: 10 mM aqueous acetic acid solution / acetonitrile = 3/97, detector: evaporative light scattering detector) and mass spectrometry, It was confirmed that THB had been produced. For example, in the sample obtained by heating the DOI fermentation broth at 132 ° C. for 20 minutes, it was revealed that about 30% of DOI was converted to THB and about 50% was converted to other dehydrated product.

  Next, a purified DOI aqueous solution (DOI concentration 70 g / L) and a DOI fermentation broth (0.2 μm MF membrane) were used under different test conditions using a tank and a heating device essentially similar to those shown in FIG. The filtered product; the DOI concentration of 59.1 g / L) was heated, and the compounds contained in the liquid after heating were analyzed to compare the results. This DOI fermentation broth was obtained by culturing Escherichia coli expressing the btrC gene in the CSL medium for 32 to 40 hours as described above. The analysis results are shown in Table 1 below.

  In Table 1, those having the same test conditions for the purified DOI aqueous solution sample and the DOI fermentation liquid sample are shown in the same row. The DOI residual rate represents what percentage of the DOI contained in the sample before heating remained after heating, and the 1,2,4-THB conversion rate was the percentage of DOI contained in the sample before heating. Is converted to 1,2,4-THB (monohydrate conversion rate is also the same). In the same experiment, the production of 1,2,3-THB was also confirmed (data not shown). For example, the description “160 ° C. 30 seconds repeated 5 times” means that heating at 160 ° C. for 30 seconds was repeated 5 times, that is, the sample was heated at 160 ° C. for a cumulative time of 2.5 minutes. means.

  From the results of Table 1, the longer the cumulative time of heating, the more the conversion amount increases (for example, comparing samples 2 to 4 of the purified DOI aqueous solution), and the higher the heating temperature, the higher the conversion efficiency. (Eg compare DOI fermentation broth samples 2 and 5, samples 3 and 6, or samples 4 and 7).

  Importantly, under the same heating conditions, the conversion of DOI to THB was achieved with significantly higher efficiency in the DOI fermentation broth compared to the purified DOI aqueous solution. That is, the THB conversion rate in the purified DOI aqueous solution sample 2 was 2.33%, while the THB conversion rate in the DOI fermentation liquid sample 5 under the same heating conditions was 27.19%. Similarly, the THB conversion rate of the purified DOI aqueous solution sample 3 was 5.23%, while the THB conversion rate of the DOI fermentation liquid sample 6 was 43.40%, and the THB conversion rate of the purified DOI aqueous solution sample 4 was The THB conversion rate of DOI fermentation liquor sample 7 was 54.66%, whereas it was 9.55%. In the DOI fermented liquor sample 7, at a high temperature of 160 ° C., a THB conversion rate of more than 50% was achieved by heating for a relatively short time of 5 minutes in total. Since similar results are obtained even when liquid media having different material components are used, it is speculated that the liquid environment itself capable of supporting bacterial growth provides some catalytic action for the THB conversion reaction.

  When the DOI fermented liquor from which the bacteria were removed by filtration was heated, the THB yield was improved more than when the DOI fermented liquor not filtered was heated, and salting out treatment was combined with the filtration. In some cases THB yields were further improved (data not shown).

  In another experiment, THB aqueous solution, a THB product-containing solution obtained by heating an aqueous DOI solution, or a product solution obtained by heating a DOI fermentation broth was subjected to solvent extraction of THB using ethyl acetate as an extraction solvent. Tested. It was confirmed that most of THB can be recovered by the first solvent extraction. The recovery of THB is improved when hydrochloric acid is added to the THB product solution to lower the pH to less than 2 before solvent extraction or when less than saturated sodium chloride is added to the THB product solution. Was observed. The yield of THB finally extracted varies depending on the conditions and the degree of the THB production reaction prior to the extraction. In the example using the THB product-containing solution obtained by heating the DOI aqueous solution at a high temperature of 175 ° C. for at least 3 hours to proceed the reaction, most of the organic compound solutes (70%) dissolved in the organic solvent layer after extraction (70 It was confirmed that THB was contained in an amount of more than 70% by weight, and in some cases, more than 90% by weight, and most of impurities remained in the aqueous layer. This result shows that the solvent extraction has a THB purification effect. THB is recovered in high yield even in the extraction using a microreactor, from the heating of the bacterial culture to the separation of THB, or from the DOI synthesis by culturing the DOI synthetic bacteria to the separation of THB via the heating of the bacterial culture. It was proved in principle that it can be carried out continuously.

An example in which DOI synthesis by culture of DOI synthetic bacteria, heating of a bacterial culture solution (DOI fermentation solution), and separation of THB by solvent extraction are continuously performed will be described below.
150 mL of DOI fermentation broth (DOI concentration: 60 g / L) obtained by culturing DOI synthetic bacteria was reacted for 2 hours at 170 ° C. to convert DOI to THB. Next, the obtained THB-containing reaction solution was filtered with a membrane filter (0.45 μm) to prepare a THB product solution ( ¹³ C-NMR spectrum is shown in the lower part of FIG. 4). Then, using a YMC mixer (Deneb Helix type), THB product solution (1 mL / min) and ethyl acetate (1 mL / min, 1.5 mL / min, 2 mL / min, and 3 mL / min, four conditions) were used. The solution was sent and the eluate was taken out (elution time 1 minute). The ethyl acetate layer of the obtained eluate was separated and concentrated under reduced pressure to obtain THB yield (all experiments were carried out in triplicate). The results are summarized in Table 2 below. The THB yield was 74.7% on average when the ratio of the THB product solution to ethyl acetate was 1: 1. The yield tended to increase as the ratio of ethyl acetate increased, and it was 86.9% on average when the ratio of the THB product solution and ethyl acetate was 1: 3. The ¹³ C-NMR spectrum of the obtained THB extracted sample is shown in FIG.

Claims (8)

A culture tank for holding a liquid medium containing bacteria that synthesize deoxyscylinosose (DOI) and culturing the bacteria to produce a bacterial culture solution;
A heating device connected to the culture tank to heat the bacterial culture at a high temperature of 80 ° C. or higher to generate a product solution containing trihydroxybenzene (THB);
System for producing trihydroxybenzene (THB).   The culture tank is connected to the heating device via a liquid supply pipe, and the heating device heats the bacterial culture solution sent from the culture tank inside the heating device. The system described in.   The system according to claim 2, wherein the culture tank is connected to the heating device via a bacterial cell removing device that removes bacterial cells of the bacteria from the bacterial culture solution.   The system according to claim 1, wherein the heating device is an integrated heating device that heats the bacterial culture solution inside the culture tank.   5. The system according to any one of claims 1 to 4, further comprising a purifier that removes solids and insolubles from the product solution.   The system according to any one of claims 1 to 5, further comprising a separation device for separating trihydroxybenzene (THB) from the product solution.   7. The system of claim 6, wherein the separator comprises a solvent extractor. The culture tank is one of a stirring device for stirring the medium, a shaking device for shaking the medium, an aeration device for aerating the medium, and a heating device for heating the medium. The system according to any one of claims 1 to 7, having the above.