Production of aminobenzoic acids

Abstract

The present invention relates to the production of aminobenzoic acid using a process having the following steps: (A) providing a fermentation broth containing aminobenzoate anions by carrying out a fermentation process in the presence of a microorganism selected from the group consisting of Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans or a mixture of two or more of the aforementioned microorganisms, and subsequently carrying out a clarification process, (B) setting the pH value of the fermentation broth containing aminobenzoate anions to 3.0 to 5.5 by adding formic acid, (C) isolating the aminobenzoic acid precipitated in step (B), and (D) introducing the mother liquor precipitated in step (C) partially or entirely into the fermentation and depleting the formate anions and formic acid contained in the mother liquor by (i) an added enzyme preparation and / or (ii) the microorganism used in step (A) and / or (iii) an additional microorganism different from the microorganism used in step (A). The aminobenzoic acid obtained by this process can be converted into products, in particular anilines, and in the case of anthranilic acid into indigo acid anhydride or anthranilate esters, which can be used as raw materials for the production of polymers such as polyurethanes or poly(anthranilamide).

Description

[0001] The research that enabled this invention was funded by the German Federal Ministry of Food and Agriculture, grant code 2221NR073X.

[0002] This invention relates to the production of aminobenzoic acid by a method comprising the following steps: (A) in a solution of Escherichia coli (Escherichia coli) Escherichia coli ), Pseudomonas putida ( Pseudomonas putida ), Corynebacterium glutamicum ( Corynebacterium glutamicum Bacillus coagulans ( Bacillus coagulans (A) In the presence of microorganisms or a mixture of two or more of the above-mentioned microorganisms, a fermentation broth containing aminobenzoate anions is provided by fermentation and subsequently clarified; (B) the pH of the fermentation broth containing aminobenzoate anions is set to 3.0 to 5.5 by adding formic acid; (C) the aminobenzoate precipitated in step (B) is removed; and (D) some or all of the mother liquor produced in step (C) is introduced into fermentation, and the formate anions and formic acid present in the mother liquor are depleted by (i) the addition of enzyme preparations and / or (ii) the microorganisms used in step (A) and / or (iii) the addition of additional microorganisms different from those used in step (A). The aminobenzoate obtained thereby can be converted into conversion products, particularly aniline, and in the case of anthranilic acid, into indomethacin or anthranilic esters, which can then be used as raw materials for the production of polymers such as polyurethane or poly(anthranilamide).

[0003] In recent years, the production of organic acids through fermentation has received particular attention. Among the organic acids obtainable through fermentation, aminobenzoic acid should also be emphasized as an economically important product. For example, aminobenzoic acid can be used to produce dyes, flavorings, crop protectants, or pharmaceuticals. Another example of the use of aminobenzoic acid is its use in the production of aniline via decarboxylation. Aniline is of particular importance as an intermediate in the production of isocyanates. The ortho-isomer of aminobenzoic acid, anthranilic acid, can also be used as a raw material for the production of poly(o-aminobenzoamide) and polyamines, as well as the corresponding polyisocyanates.

[0004] The fermentation production of aminobenzoic acid is known in principle in the prior art; see, for example, International Patent Application WO2015 / 124687 A1 (which describes a two-stage production of aniline via an-aminobenzoic acid as an intermediate) and the references cited therein. The fermentation process is carried out in an aqueous culture medium, and in the case of producing aminobenzoic acid, a mixture of aqueous products is typically provided, particularly in the range of 10.0 g / L to 100 g / L. Fermentation liquid ).

[0005] Of particular importance is the ortho-isomer of aminobenzoic acid (annaminobenzoic acid). In bacterial and yeast metabolism, annaminobenzoic acid is formed in the shikimic acid pathway as a natural intermediate in tryptophan synthesis. In the biotechnological production of annaminobenzoic acid, its conversion in the metabolic pathway is reduced or inhibited to achieve accumulation in the fermentation medium. The international patent applications WO 2015 / 124686 A1 and WO 2015 / 124687 A1, which have already been mentioned, describe this concept for the biotechnological production of annaminobenzoic acid and its subsequent catalytic conversion to aniline. One possible recombinant microorganism described is the use of bacteria from the Corynebacterium or Pseudomonas families. A more recent application (WO 2017 / 102853 A1) describes the use of yeast.

[0006] However, para-aminobenzoic acid (p-aminobenzoic acid) is also of interest. P-aminobenzoic acid can be synthesized in bacteria and yeast via the intermediate cladonic acid, which is formed as an intermediate in the shikimic acid pathway. The cladonic acid is first enzymatically converted to 4-amino-4-deoxycladonic acid, and then converted to p-aminobenzoic acid via a second enzymatic reaction. The concept of producing aniline via the intermediate p-aminobenzoic acid biotechnology is described in international application US 2016 / 068876 A1. Another possible recombinant microorganism described herein specifically utilizes bacteria from the Corynebacterium family.

[0007] The fermentation production of aminobenzoic acid provides an aqueous product stream. When fermentation is carried out at a pH significantly above the isoelectric point (the typical pH range when the microorganism used is bacteria), the valuable aminobenzoic acid product exists primarily or entirely in anionic form (aminobenzoate anion). To neutralize the aminobenzoic acid formed during fermentation, an alkali, particularly sodium hydroxide, is typically added as a pH control agent. After the usual fermentation treatment steps, the valuable aminobenzoic acid product can precipitate (crystallize) in a solid, electrically neutral form by adjusting the pH to near or equal to the isoelectric point. The aminobenzoic acid can then be separated, for example, by filtration. The filtered product is initially in a high-water-content state (“…”). Pulp materialThe product is then, for example, washed and dried if necessary (depending on the intended use), or absorbed in a solvent such as aniline or 1-dodecanool (see WO 2015 / 124687 A1). To adjust the pH during crystallization, inorganic acids, particularly hydrochloric acid, are typically used. This is described, for example, in international application WO 2017 / 085170 A1, which relates to a method for producing aminobenzoic acid or aminobenzoic acid derivatives by fermenting a suitable feedstock under the influence of suitable microorganisms to obtain a fermentation broth containing aminobenzoic acid esters and / or aminobenzoic acid, with particular emphasis on obtaining aminobenzoic acid from the fermentation broth by single-stage acid treatment crystallization in the presence of seed crystals. However, the use of inorganic acids in crystallization inevitably leads to the formation of corresponding salts, particularly sodium chloride. This salt load is a significant wastewater pollutant and is therefore economically detrimental.

[0008] As an alternative to using inorganic acids, in a specific configuration for the fermentation production of aminobenzoic acid, carbon dioxide can also be introduced under pressure to crystallize aminobenzoic acid (crystallization with "carbon dioxide"). This method is described in WO 2019 / 234092A1 (also published as US 2021 / 0222215 A1). In this method, fermentation is carried out in the presence of calcium salts, wherein (I) some aminobenzoic acid present in the fermentation broth binds to insoluble calcium aminobenzoate, to the greatest extent possible due to solubility equilibrium, then (II) the insoluble calcium aminobenzoate is separated, either on its own or in a mixture with the microorganisms used in the fermentation, and converted to a water-soluble form by separating insoluble calcium salts different from calcium aminobenzoate [ion exchange], then (III) aminobenzoic acid is precipitated [crystallization] by introducing carbon dioxide under pressure into an aqueous solution from which the precipitated calcium salts have been removed. The advantages of this method are that it reduces the concentration of dissolved aminobenzoic acid in the fermentation reactor, most of which may crystallize with carbon dioxide, and it also reduces the use of alkali in fermentation.

[0009] Removing aminobenzoic acid through filtration leaves a mother liquor containing a significant residual concentration of the acid (depending on its solubility under specific conditions). Therefore, dissolved aminobenzoic acid should be removed quantitatively as much as possible before treating it as wastewater. Furthermore, the mother liquor often contains dissolved residues of usable culture medium components, including carbon or nitrogen sources that were not fully converted to aminobenzoic acid or biomass during fermentation. The effort required to remove these usable culture medium components from the mother liquor typically outweighs the economic benefits, meaning that wastewater treatment is preferred in existing technologies.

[0010] To obtain aminobenzoic acid from dilute aqueous solutions, various concepts have been developed in the prior art, as described on pages 3-5 of WO 2023 / 117756 A1. As described therein, none of these concepts is without drawbacks. WO 2023 / 117756A1 itself proposes extracting an aqueous mother liquor containing dissolved aminobenzoic acid from an alkanol having 8 to 12 carbon atoms, obtained by the method described therein, followed by alkaline or acidic back-extraction to obtain the aminobenzoic acid dissolved in the alkanol phase as an aqueous solution of aminobenzoate, and finally precipitating it from the salt solution by adjusting the pH to 3.0 to 4.7. Although C8-C... 12 Using alkanols as extractants in conjunction with alkaline or acidic back-extraction offers a good compromise between maximizing the extraction of aminobenzoic acid from the mother liquor (for maximum yield) and obtaining a mother liquor with minimal admixture (for simplified wastewater treatment / disposal). However, this method is not without its drawbacks. The need to extract all of the mother liquor necessitates considerable effort. More seriously, this application does not provide a fundamental solution to the problem of high salt loading in wastewater. In the aforementioned application WO 2019 / 234092 A1, it is suggested that the mother liquor remaining after the removal of aminobenzoic acid precipitated in carbon dioxide crystallization be recycled to an ion exchange step. Furthermore, it is suggested that additional aminobenzoic acid be crystallized from the fermentation aqueous solution remaining after the removal of calcium aminobenzoate produced during fermentation by adding an inorganic acid. In this case, the additional mother liquor remaining after removing the precipitated aminobenzoic acid can undergo an adsorption step to obtain the residual aminobenzoic acid dissolved therein, and the adsorbed aminobenzoic acid can be recovered through a subsequent desorption step. Depending on the pH at which desorption occurs, the resulting eluent can be recycled to the ion exchange step (desorption at pH 6.0 to 11.0) or post-crystallized by adding alkali (desorption at pH less than 3.0). WO 2019 / 234092 A1 does not disclose the recycling of mother liquor (regardless of source) into fermentation.

[0011] Therefore, further improvements are needed in the fermentation production of aminobenzoic acid. In particular, it is desirable to reduce salt pollution of wastewater and yield loss caused by mother liquor.

[0012] In view of this need, the present invention provides a method for producing aminobenzoic acid, the method comprising the following steps: (A) Provides a fermentation broth containing aminobenzoate anions, comprising: (A.1) fermenting fermentable carbon and nitrogen compounds at pH > 5.5 in the presence of microorganisms selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans, or a mixture of two or more of the above microorganisms; and (A.2) removing the microorganisms; (B) The pH of the fermentation broth containing aminobenzoate anions is set to 3.0 to 5.5, preferably 3.5 to 4.5, more preferably 3.8 to 4.2, and most preferably 4.0 by adding formic acid, in order to precipitate aminobenzoic acid; (C) Remove the aminobenzoic acid precipitated in step (B) to obtain a mother liquor containing formate anions and formic acid; and (D) 55.0% to 100% of the total mother liquor (containing formate anions and formic acid) generated in step (C) is optionally introduced into the fermentation according to step (A.1) after treatment (e.g., particularly aseptic filtration and / or heat inactivation), and the formate anions and formic acid are depleted in the following manner. (i) Added enzyme preparations, and / or (ii) Microorganisms used in step (A) and / or (iii) Additional microorganisms different from those used in step (A) (By oxidizing formate anions and formic acid to carbon dioxide and / or by using them as substrates for biomass production and / or by using them as substrates for biomass production via microorganisms from step (A) and / or by using them as substrates for biomass production via additional microorganisms).

[0013] It has been discovered, quite surprisingly, that the use of formic acid in the crystallization of aminobenzoic acid results in the formation of a mother liquor that can be recycled back into fermentation without the accumulation of formate anions or formic acid. When formic acid (instead of strong inorganic acids commonly used in the prior art, such as hydrochloric acid, in particular) is used as the crystallizing agent for aminobenzoic acid, a mother liquor containing formate anions formed by the protonation of aminobenzoic acid anions is produced. Furthermore, the mother liquor also contains additional free formic acid because, compared to HCl, formic acid does not completely dissociate under current conditions to form H₃O. + Ions. For simplicity, the following description refers to "ions". Mother liquor containing formate anions This wording includes the presence of free formic acid. Formic acid, whether in the form of formate anions or free acid, is oxidized to carbon dioxide during fermentation, binds to the target fermentation product, aminobenzoate anions, and / or is metabolized to build biomass. This is achieved by the microorganisms used in step (A), or, if they cannot or cannot do so adequately, by the addition of enzymes or additional microorganisms different from those used in step (A) that are capable of doing so. These options can also be combined with each other. Details on how to provide suitable microorganisms and enzymes will be given below.

[0014] Equally surprising is the finding that, despite the relatively low pH of the mother liquor containing formate anions, ranging from 3.0 to 5.5 (see step (B) above), recycling this mother liquor into fermentation does not imply the need to add more alkali to observe the pH required for fermentation. On the contrary, the trend has been found to be that even less alkali is needed to adjust the pH, ideally none at all. Without being bound by theory, it is believed that the degradation of formate during fermentation occurs at least partially with the release of carbon dioxide from the fermentation broth, and this "carbon dioxide" release leads to an increase in pH, which compensates for the initial decrease in pH caused by the introduction of acidic mother liquor, and ideally, even additionally compensates for the decrease in pH caused by the formation of aminobenzoic acid.

[0015] Furthermore, recycling the mother liquor containing formate anions results in additional dissolved components, such as unreacted carbon or nitrogen sources and unreacted nutrients, such as salts, trace elements, and vitamins, being recycled back into the fermentation process. Additionally, a portion of the aminobenzoic acid still dissolved in the mother liquor is recycled, meaning that the extra effort required to obtain dissolved aminobenzoic acid from the mother liquor is not applicable to the recycled portion.

[0016] All pH values ​​in the context of this invention relate to the temperature at which the corresponding steps (e.g., steps (A.1) or (B)) are performed, and can be simply measured using a glass electrode.

[0017] First, various possible embodiments of the present invention. Brief Overview : In the first embodiment of the invention—which can be combined with all other embodiments—water, particularly evaporated water, is removed (partially) after step (A.2) and before step (B) or after step (B) and before step (C).

[0018] In a second embodiment of the invention—which can be combined with all other embodiments—the nitrogen-containing compound is selected from ammonia, ammonia water, (at least one) ammonium salt, soybean protein, urea, or a mixture of two or more of the above-mentioned nitrogen-containing compounds.

[0019] In a third embodiment of the invention—which can be combined with all other embodiments—the fermentable carbonaceous compound is selected from starch hydrolysates, alkali metal formates or ammonium formates, sugarcane juice, beet juice, hydrolysates of lignocellulose raw materials, or mixtures of two or more of the above-mentioned carbonaceous compounds.

[0020] In a fourth embodiment of the invention—which can be combined with all other embodiments, as long as they do not preclude the use of additional microorganisms different from those used in step (A)—the additional microorganisms are selected from Pichia pastoris (… Pichia pastoris ), Hansenula polymorpha ( Hansenula polymorpha (or a mixture of both)

[0021] In the fifth embodiment of the invention—which can be combined with all other embodiments—a pH of 5.6 to 11, preferably 6.0 to 8.0, more preferably 6.6 to 8.0, is observed in step (A.1).

[0022] In a sixth embodiment of the invention—which can be combined with all other embodiments—formic acid is used as a mixture of formic acid and water or as anhydrous formic acid, wherein the mass concentration of formic acid in the mixture is at least 20% based on the total mass of the mixture, preferably 60% to 98%, more preferably 70% to 95%.

[0023] In a seventh embodiment of the invention—which can be combined with all other embodiments except those limited to forming m-aminobenzoic acid or para-aminobenzoic acid—o-aminobenzoic acid is produced.

[0024] In the eighth embodiment of the invention—which can be combined with all other embodiments except those limited to forming anthraquinone or metaquinone—paraquinone is produced.

[0025] In the ninth embodiment of the invention—which is a specific configuration of the seventh embodiment—the anthranilic acid obtained in step (C) is converted into poly(anthranilamide).

[0026] In the tenth embodiment of the invention—which is another specific configuration of the seventh embodiment—the anthranilic acid obtained in step (C) is converted into an anthranilic acid derivative selected from anthranilic benzoyl halides, indigo anhydride, or mixtures thereof, and the anthranilic acid derivative is reacted with a polyol to form a polyamine.

[0027] In the eleventh embodiment of the invention—which is a specific configuration of the tenth embodiment—polyamine is phosgenated to form polyisocyanate.

[0028] In the twelfth embodiment of the invention—which can be combined with all other embodiments except those that do not involve the formation of aniline from aminobenzoic acid, and particularly advantageously with the seventh embodiment—the aminobenzoic acid from step (C) is converted into aniline, eliminating carbon dioxide.

[0029] In the thirteenth embodiment of the invention—a specific configuration of the twelfth embodiment—aniline reacts with formaldehyde to form methylene diphenylene diamine and polymethylene polyphenylene polyamine.

[0030] In the fourteenth embodiment of the invention—which is a specific configuration of the thirteenth embodiment—methylene diphenylene diamine and / or polymethylene polyphenylene polyamine are phosgenated to form methylene diphenylene diisocyanate and / or polymethylene polyphenylene polyisocyanate.

[0031] In the fifteenth embodiment of the invention—a specific configuration of the fourteenth embodiment—methylene diphenyl diisocyanate and / or polymethylene polyphenyl polyisocyanate react with a polyol to form a polyurethane.

[0032] In the sixteenth embodiment of the invention—which is another specific configuration of the twelfth embodiment—aniline is converted into an azo compound.

[0033] In the seventeenth embodiment of the invention—which is a specific configuration of the twelfth to sixteenth embodiments—the carbon dioxide formed during the conversion to aniline is converted to formic acid, and the formic acid thus obtained is used in step (B).

[0034] In the eighteenth embodiment of the invention—which can be combined with all other embodiments—the carbon dioxide that may be formed during fermentation in step (A.1) (especially by the oxidation of formate anions and / or formic acid) is converted into formic acid, and the formic acid thus obtained is used in step (B).

[0035] In the nineteenth embodiment of the invention—which is a specific configuration of the seventeenth and eighteenth embodiments—the conversion to formic acid is achieved by hydrogenation of carbon dioxide or acidic electrolysis.

[0036] In the twentieth embodiment of the invention—which can be combined with all other embodiments—in step (D), a first portion of the mother liquor is mixed with an additional carbon-containing compound, an additional nitrogen-containing compound, at least one compound for reducing foaming during fermentation, such as, in particular, polypropylene glycol, at least one inorganic salt, such as, in particular, potassium phosphate or calcium chloride, at least one trace element, such as, in particular, ferric sulfate, manganese sulfate, copper sulfate, zinc sulfate or nickel chloride, or a mixture of two or more of the above compounds.

[0037] In the twenty-first embodiment of the invention—which can be combined with all other embodiments—in step (D), less than 100%, particularly up to 99.5%, of the total mother liquor produced in step (C) is introduced into the fermentation according to step (A.1), and the portion of the mother liquor not introduced into the fermentation is depleted of aminobenzoic acid, followed by wastewater treatment.

[0038] In the twenty-second embodiment of the invention—which is a specific configuration of the twenty-first embodiment—the depletion of aminobenzoic acid comprises extraction with an organic solvent and / or adsorption on an adsorbent, followed by desorption.

[0039] In the twenty-third embodiment of the invention—which is a specific configuration of the twenty-second embodiment—the organic solvent comprises an alkanol having 8 to 12, preferably 9 to 11, carbon atoms, particularly 1-dodecanool, and wherein the adsorbent is activated carbon or a polymer adsorbent, particularly an adsorbent based on polystyrene and / or polydivinylbenzene.

[0040] In the twenty-fourth embodiment of the invention—which can be combined with all other embodiments as long as they do not exclude the use of an enzyme preparation—the enzyme preparation comprises formate dehydrogenase and / or formate oxidase.

[0041] In the twenty-fifth embodiment of the invention—which is a specific configuration of the twenty-fourth embodiment—formate dehydrogenase belongs to EC 1.17.1.9 and formate oxidase belongs to EC 1.2.3.1.

[0042] The following text will To elaborate in more detail The above briefly outlines the embodiments and further possible configurations of the invention. All the above embodiments and the further configurations of the invention described below can be combined with each other and in combination as needed, unless it is obvious to those skilled in the art, based on the context, that the opposite is not explicitly stated.

[0043] Provide a fermentation broth containing aminobenzoate anions (step (A)) Step (A) of the method includes at least step (A.1), Fermentation of carbonaceous compounds in the presence of microorganisms and nitrogen-containing compounds, and (A.2) Remove microorganisms Fermentation takes place in a reaction apparatus provided for this purpose, namely... Fermentation reactor The fermentation is carried out in the fermentation reactor. The reaction mixture present in the reactor is called the fermentation broth. Fermentation in step (A.1) is carried out in such a way that the pH of the fermentation broth is greater than 5.5, preferably in the range of 5.6 to 11, more preferably in the range of 6.0 to 8.0, and most preferably in the range of 6.6 to 8.0. If necessary, the pH can be controlled by adding alkali, particularly by adding ammonia water or gaseous ammonia, aqueous potassium hydroxide solution or aqueous sodium hydroxide solution. At these pH values, aminobenzoic acid is mainly or even entirely in its anionic form (aminobenzoate anion, H2NC6H4COO-); therefore, the present invention relates to... Contains aminobenzoate anion fermentation broth (The type of counter ion depends on the exact conditions; in particular, it could be Na.) + K + and / or NH4+ It has been found, quite surprisingly, that in the method according to the invention, the addition of alkali for pH control can be reduced, and ideally can be omitted entirely, due to the recycling of the mother liquor containing formate anions (except in possible special cases, such as when starting the method).

[0044] The preferred microorganisms for step (A.1) are prokaryotes (especially bacteria). Examples of suitable microorganisms include *Escherichia coli*, *Pseudomonas putida*, *Corynebacterium glutamicum*, and *Bacillus coagulans*. *Escherichia coli*, *Pseudomonas putida*, and / or *Corynebacterium glutamicum* are preferred. A mixture of different microorganisms may be used, but the use of a single species is preferred. *Corynebacterium glutamicum*, particularly ATCC 13032, is especially preferred. In this regard, particular reference is made to patent applications WO 2015 / 124686 A1 and WO 2015 / 124687 A1, which describe fermentation methods using bacteria (see, for example, WO 2015 / 124687 A1, page 15, lines 8-16, line 30, Example 1 (page 29, lines 4-26), Example 3 (particularly page 34, lines 10-18), and Example 4 (particularly page 55, lines 9-31). Specifically, the bacteria used are those capable of converting fermentable carbonaceous compounds into aminobenzoate anions in the presence of a suitable nitrogen source, and these anions are not directly consumed in intracellular biochemical processes. As a result, the aminobenzoate anions are enriched in the cells and ultimately transferred to the fermentation broth.

[0045] In principle, there are two pathways available for obtaining this prokaryote, and these pathways can also be combined in preferred configurations: (i) It can increase the enzymatic reactions in the aminobenzoic acid metabolic pathway in prokaryotic cells, making the production of aminobenzoic acid faster than its consumption.

[0046] (ii) Subsequent reactions that convert aminobenzoic acid into other metabolites or products (e.g., tryptophan) can be reduced or shut down, resulting in the enrichment of aminobenzoic acid in cells.

[0047] Methods for obtaining prokaryotes with the aforementioned characteristics are known from the prior art. Suitable prokaryotes can be identified, for example, by screening for mutants that secrete aminobenzoic acid into the surrounding culture medium. However, it is preferable to specifically modify the key enzymes using genetic engineering methods. Using conventional genetic engineering methods, gene expression and enzyme activity can be enhanced, reduced, or even completely inhibited. Recombinant strains are the result. For particularly preferred ortho isomers, preferred embodiments are described below (for para isomers, see, for example, T. Kubota et al.). Metabolic Engineering 2016, 38, 322 –330 (“ Production of para-aminobenzoate by genetically engineered Corynebacterium glutamicum and non-biological formation of an N-glucosyl byproduct "[1]): More preferably, prokaryotes capable of converting fermentable carbonaceous compounds into aminobenzoic acid in the presence of nitrogen-containing compounds contain modifications to the activity of an-aminobenzoate phosphoribosyltransferase, which reduces the activity of said enzyme. Due to this modification, the conversion of an-aminobenzoate to N-(5-phosphate-D-ribosyl)-an-aminobenzoate is reduced or completely inhibited. This leads to the enrichment of aminobenzoic acid in cells. The expression "an-aminobenzoate phosphoribosyltransferase activity" herein refers to the enzyme activity catalyzing the conversion of an-aminobenzoate to N-(5-phosphate-D-ribosyl)-an-aminobenzoate.

[0048] In Corynebacterium glutamate, the activity of anthranilate phosphoribosyltransferase is determined by... trpD The genes (cg3361, Cgl3032, NCgl2929) encode this. In the case of *Pseudomonas putida*, the encoding is... trpDC within the manipulator trpD Gene (PP_0421) was achieved.

[0049] The reduction of anthranilate phosphoribosyltransferase activity can be achieved in principle through three methods: (i) It can modify the regulation of gene expression of anthranilate phosphoribosyltransferase activity, thereby reducing or inhibiting gene transcription or subsequent translation.

[0050] (ii) The nucleic acid sequence of the anthranilate phosphoribosyltransferase active gene can be modified so that the enzyme encoded by the modified gene has a lower specific activity.

[0051] (iii) The natural gene for anthranilate phosphoribosyltransferase activity can be replaced by a different gene derived from a different organism and encoding an activity greater than that of the natural gene described above (e.g., TRP4, ...). trpD or trpDC The enzyme has a lower activity than the anthraquinone phosphoribosyltransferase.

[0052] In a preferred embodiment of the invention, *Corynebacterium glutamicum*, preferably *Corynebacterium glutamicum* ATC13032, is used as a microorganism to provide a fermentation broth containing aminobenzoate anions. They preferably contain the modifications defined below. Further details regarding this strain and its metabolic capabilities are disclosed in WO 2023 / 111055.

[0053] (i) Compared to the corresponding wild type, the activity of an-aminobenzoic acid phosphoribosyltransferase is reduced. In one embodiment, there is no corresponding enzyme activity. Genetic modifications that enable this are well known. These include deletion of the relevant gene or modification of the coding region of the gene, such that the expressed enzyme is truncated or inactivated for other reasons. In an alternative embodiment, the activity of the enzyme has been reduced compared to the activity present in the wild type, although some residual activity must still be present. This residual activity is preferably 10% to 60% of the natural activity in Corynebacterium glutamicum ATCC13032, more preferably 20% to 50%. This is preferably achieved by reducing the activity of an-aminobenzoic acid phosphoribosyltransferase (...) compared to the wild type. trpD This is achieved by inhibiting gene expression, although expression is not completely suppressed. This is preferably achieved by using a promoter sequence with lower transcriptional activity compared to the endogenous promoter sequence, or by altering the ribosome binding site. trpD The distance of the gene start codon, or by changing the start codon itself. In a preferred embodiment of the invention, the activity of anthranilate phosphoribosyltransferase is mediated by endogenous anthranilate phosphoribosyltransferase (… trpD The deletion or inactivation of the gene, or its replacement with an anthranilate phosphoribosyltransferase gene having a modified ribosome binding site and optionally a modified start codon, can reduce the concentration of the gene, as defined in SEQ ID NO. 2 or 6, preferably SEQ ID NO. 6. The amino acid sequence of the anthranilate phosphoribosyltransferase preferably corresponds to that of the endogenous anthranilate phosphoribosyltransferase, more preferably as defined in SEQ ID NO. 7 or a variant thereof.

[0054] (ii) Increased shikimate kinase activity. This is preferably achieved by increasing the expression of the corresponding enzyme. In one embodiment of the invention, activity is increased by enhancing the expression of the endogenous shikimate kinase gene or a variant thereof as defined in SEQ ID NO. 8. In another preferred embodiment, this is achieved by the expression of exogenous shikimate kinase, preferably as defined in SEQ ID NO. 9 or a variant thereof. Enhancement of gene expression can be achieved by any method known to those skilled in the art, particularly by introducing two or more copies of the corresponding gene into a microorganism or by expressing the endogenous enzyme using a stronger promoter. A particularly preferred promoter for expressing exogenous genes or enhancing the expression of endogenous genes is P as defined in SEQ ID NO. 10. tuf .

[0055] (iii) Enhanced activity of 3-phosphoshikimate-1-carboxyvinyltransferase and branched acid synthase. Preferably, these enzymes have the amino acid sequences defined in SEQ ID NO. 11 or a variant thereof and SEQ ID NO. 12 or a variant thereof. This is preferably achieved by introducing an additional copy of the gene encoding these enzymes into the microorganism. tuf Promoters are preferred for controlling expression.

[0056] (iv) It is advantageous, but not essential, to have 3-deoxyarabinohepenolate 7-phosphate synthase (DAHP synthase) which has "feedback resistance," meaning it is not inhibited by its product or any product formed from it. Preferably, the enzyme has the amino acid sequence defined in SEQ ID NO. 13 or a variant thereof.

[0057] In this regard, those skilled in the art will know that, based on the known metabolic pathway by which *Corynebacterium glutamicum* produces anthranilic acid, it is possible to introduce further modifications to increase the efficiency of the aforementioned strains.

[0058] In another preferred embodiment of the invention, as disclosed in WO 2022 / 090363, cells of Escherichia coli, preferably Escherichia coli K12, are used as the microorganism providing the fermentation broth containing aminobenzoate anions.

[0059] Escherichia coli strains expressing anthraquinone phosphoribosyltransferase (TrpD) and glutamine aminotransferase (TrpG) are particularly preferred.

[0060] Preferably, the TrpG domain defined by amino acids 3 to 196 of SEQ ID NO. 14 (TrpGD from Escherichia coli) or a variant thereof is preferred. Also preferred are those from Bacillus subtilis (…). Bacillus subtilis TrpG (SEQ NO.15) from Salmonella Typhimurium ( Salmonella typhimurium The TrpG domain of TrpGD (from amino acids 3 to 196 of SEQ ID NO. 16), from copper-loving bacteria ( Cupriavidus necator TrpG (SEQ ID NO. 17), TrpG (SEQ ID NO. 18) from Corynebacterium glutamicum, or any variant of the above polypeptides.

[0061] The preferred embodiment is the TrpD domain defined by amino acids 202 to 531 of SEQ ID NO. 14 (TrpGD from Escherichia coli) or a variant thereof. Also preferred are TrpD from Bacillus subtilis (SEQ NO. 19), the TrpD domain of TrpGD from Salmonella Typhimurium (amino acids 202 to 531 of SEQ ID NO. 16), TrpD from Copper-loving Bacteria (SEQ ID NO. 20), TrpD from Corynebacterium glutamicum (SEQ ID NO. 21), or any variant of the above polypeptides.

[0062] When expressing TrpG and TrpD separately, it is preferable that the expression of TrpD is lower than that of TrpG. Preferably, the expression is reduced by at least 10%, more preferably by at least 20%, even more preferably by at least 40%, and most preferably by at least 60%. However, it is particularly preferable to maintain the minimum expression level of TrpD at at least 5% of the expression level of TrpG. The expression of the genes encoding the above proteins is preferably determined at the mRNA level by quantitative TR-PCR. To achieve this different expression of the two peptides, it is preferable to introduce the genes encoding them into the cells in the form of different expression cassettes, under the control of promoters of different strengths. Alternatively, the corresponding genes can be introduced at different copy numbers. In this case, the same promoter can be used for the expression of both peptides.

[0063] In yet another embodiment, cells of *Pseudomonas putida*, preferably *Pseudomonas putida* KT2440, are used as the microorganism providing the fermentation broth containing the aminobenzoate anion. Example 4 of WO 2015 / 124687 describes a genetic modification that enables the bacteria to synthesize anthranilic acid.

[0064] In step (A), the depletion of formate anions and formic acid is preferably achieved by microorganisms or enzyme preparations. Microorganisms can also be combined with enzyme preparations.

[0065] In one embodiment of the invention, the microorganisms used to deplete formate anions and formic acid are the same as those used to provide a fermentation broth containing aminobenzoate anions. In many cases, these microorganisms already naturally express the necessary enzymes, i.e., without any further genetic modifications. For example, formate dehydrogenases of some Escherichia coli strains are known (Sawers, 1994, "The hydrogenases and formate dehydrogenases of Escherichia coli “Antonie van Leeuwenhoek, 66: 57-88.” Formate dehydrogenase has been described for use in Corynebacterium glutamicum (Witthof et al., 2012, “ Corynebacterium glutamicum Harbours a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate (Microbiology, 158: 2428-2439) and *Pseudomonas harbours* (Roca et al., 2009, “Redundancy of Enzymes for Formaldehyde Detoxification in…”). Pseudomonas putida (Journal of Bacteriology, 191: 3367-3374). If the microorganisms used lack enzymes that degrade formate anions or formic acid, or if the natural enzyme activity is insufficient, known genetic engineering methods can be used to increase the corresponding abilities of the microorganisms. This can be achieved by (i) introducing exogenous genes, especially those encoding formate dehydrogenases or oxidases, into the microorganisms; (ii) introducing additional copies of existing natural genes for the corresponding enzymes; or (iii) replacing the natural promoter of the natural gene for the corresponding enzyme with a stronger promoter.

[0066] In another embodiment, additional microorganisms unsuitable for providing aminobenzoic acid are used. In this case, the fermentation broth contains at least two types of microorganisms: (i) microorganisms for providing aminobenzoic acid through fermentation, and (ii) microorganisms solely for depleting formate anions and formic acid (“additional microorganisms”). In one embodiment, the additional microorganisms for depleting formate anions and formic acid are those that already naturally express sufficient amounts of the corresponding enzymes. In another embodiment, the additional microorganisms used are those whose corresponding enzyme activities have been produced or enhanced by the methods of the embodiments defined in the preceding paragraph. If additional microorganisms are used such that the fermentation production of aminobenzoic acid and the depletion of formate anions and formic acid are separate, the additional microorganisms do not necessarily need to still be able to reproduce. Therefore, in one embodiment of the invention, additional microorganisms that are no longer able to reproduce can be used. For example, this can be achieved by using auxotrophic strains that have not been compensated for in the fermentation for the production of aminobenzoic acid. Alternatively or additionally, heating is also possible, provided that the additional microorganisms are sufficiently temperature-stable.

[0067] The term "enzyme preparation" refers to a composition containing (at least) formate dehydrogenase and / or (at least) formate oxidase and / or (at least) formate hydrogen lyase, preferably containing formate dehydrogenase and / or formate oxidase. Preferred formate dehydrogenases and oxidases are described below in this application. According to the invention, the formate dehydrogenase or oxidase need not be in pure form. Thus, in one embodiment of the invention, the enzyme preparation is a crude extract of cells, preferably microorganisms, expressing the relevant enzyme. In another embodiment, the enzyme preparation comprises or consists of at least one purified formate oxidase or dehydrogenase.

[0068] To deplete formate anions or formic acid, formate dehydrogenases, preferably EC 1.17.1.9, or formate oxidases, preferably EC 1.2.3.1, are preferred.

[0069] In this document, formate dehydrogenases and variants thereof as defined in SEQ ID NO. 22 or 23 are preferred. Formate oxidases are enzymes and variants thereof as defined in SEQ ID NO. 24 or 25.

[0070] In this application, "variant" is understood to refer to a polypeptide obtained by adding, deleting, or substituting up to 20%, preferably up to 15%, more preferably up to 10%, and most preferably up to 5% of an amino acid present in a specific polypeptide. The above modifications can, in principle, be performed continuously or discontinuously at any desired point in the polypeptide. However, they are preferably produced only at the N-terminus and / or C-terminus of the polypeptide. Amino acid substitutions are preferably conservative substitutions, i.e., wherein the modified amino acid has residues with chemical properties similar to those present in the unmodified polypeptide. Therefore, it is more preferable to exchange an amino acid with a basic residue for an amino acid with a basic residue, to exchange an amino acid with an acidic residue for an amino acid with the same acidic residue, to exchange an amino acid with a polar residue for an amino acid with a polar residue, and to exchange an amino acid with a nonpolar residue for an amino acid with a nonpolar residue. The specific enzyme activity of a variant of one of the above-defined polypeptides is preferably at least 80% of the specific activity of the unmodified polypeptide. Enzyme assays for verifying the above-described enzyme activity can be found in the literature by those skilled in the art.

[0071] There are three types of aminobenzoic acid. Heterogeneous forms(o-aminobenzoic acid, m-aminobenzoic acid, and p-aminobenzoic acid). In principle, this method can be applied to all three isomers, whether in pure isomer form or a mixture of different isomers. However, it is preferred to produce o-aminobenzoic acid or p-aminobenzoic acid, especially in pure isomer form. Particularly preferred is the production of o-aminobenzoic acid, especially in pure isomer form. In this respect, "pure isomer" means that, based on all present aminobenzoic acid isomers, the molar percentage of the desired aminobenzoic acid isomer is at least 99.0 mol%, preferably at least 99.9 mol%, more preferably 100 mol%. As is known in the art, the formation of the desired isomer can be controlled enzymatically. For example, via the shikimic acid pathway, the branched acid can be enzymatically converted to o-aminobenzoate (= anion of o-aminobenzoic acid). Alternatively, there are also enzymatic reactions of branched acids to produce p-aminobenzoate (= anion of p-aminobenzoic acid).

[0072] Regardless of the microorganism used or the isomer required, the fermentation broth at the start of fermentation in step (A.1) contains recombinant cells of the microorganism used and at least one fermentable carbonaceous compound (and at least one nitrogenous compound as a nitrogen source). Preferably, the fermentation broth also contains other components selected from buffer systems, inorganic nutrients, amino acids, vitamins, and other organic compounds required for the growth or internal metabolism of the recombinant cells. The fermentation broth is water-based. After the fermentation process begins, the fermentation broth also contains aminobenzoate anions, i.e., the target fermentation product.

[0073] As already mentioned, in the context of this invention, fermentable carbonaceous compounds are understood to mean any organic compound or mixture of organic compounds that can be used to produce aminobenzoate anions via recombinant cells of the microorganisms used. The production of aminobenzoate anions can be carried out under aerobic or anaerobic conditions. Production is preferably carried out in the presence of oxygen, particularly by introducing an oxygen-containing gas such as air.

[0074] This paper prefers fermentable carbon-containing compounds that can also serve as energy and carbon sources for the recombinant cell growth of the microorganisms used. Suitable options include starch hydrolysates, alkali metal or ammonium formate, sugarcane juice, beet juice, and / or hydrolysates of lignocellulose raw materials, with starch hydrolysates, sugarcane juice, beet juice, and / or hydrolysates of lignocellulose raw materials being preferred. The nitrogen source used is preferably ammonia, ammonia water, (at least one) ammonium salt, soybean protein, and / or urea.

[0075] In one embodiment, step (A.1) is carried out continuously, i.e., the reactants are continuously fed into the fermentation reactor, and the product is continuously removed from the fermentation reactor. In the simplest case, the product continuously removed from the fermentation reactor is an aqueous fermentation broth containing microorganisms and aminobenzoate anions. Microorganisms can be removed outside the fermentation reactor by separation methods known in the art, such as, in particular, filtration, centrifugation, or sedimentation. However, it is also conceivable to use known separation methods (especially filtration) to retain microorganisms in the fermentation reactor and extract the product containing aminobenzoate anions. clarify Fermentation broth. Therefore, in this embodiment, the step of removing microorganisms (A.2) has already been carried out in the fermentation reactor itself.

[0076] In another implementation, step (A.1) is in Fermentation cycle The process is carried out in a discontinuous manner (“batch mode”). The fermentation cycle preferably includes the initial addition or supplementation of microorganisms to the culture medium, the initial addition and / or supplementation of nutrients, the accumulation of microorganisms, the formation of the desired product, the aminobenzoate anion, and the complete or partial evacuation of the reactor at the end of fermentation. In a variant of the batch operation mode (referred to as the “feed-batch mode”), the reactants are optionally fed into the fermentation reactor (continuously or discontinuously [i.e., batch]) after pre-sterilization by filtration or heat treatment, provided the reactor volume allows, without any product (excluding any gaseous components discharged into the exhaust system through the fermentation reactor connections) being discharged from the fermentation reactor. The reaction is stopped after adding the maximum possible amount of reactants, and the product mixture is removed from the fermentation reactor. In the case of a discontinuous process, it is preferable to clarify the fermentation broth outside the fermentation reactor, particularly by filtration, centrifugation, or sedimentation. If necessary, the fermentation broth may be sterilized by filtration or heat treatment before recycling.

[0077] The removed microorganisms (biomass) can be recycled back into the fermentation process, minus any discharged portion.

[0078] Regardless of the exact operating mode, the fermentation reactor preferably includes devices for measuring important process parameters, such as temperature, pH, substrate and product concentrations, dissolved oxygen content, and cell density of the fermentation broth. Particularly preferably, the fermentation reactor includes devices for adjusting at least one (preferably all) of the above-mentioned process parameters.

[0079] Suitable fermentation reactors are stirred tank reactors, membrane reactors, or circulating reactors. For aerobic and anaerobic fermentation, stirred tank reactors and circulating reactors (preferably airlift reactors, where liquid circulation in the reactor is achieved by jetting) are particularly preferred.

[0080] In addition to clarification (step (A.2)), the fermentation broth from step (A.1) may undergo further pretreatment steps before being fed into step (B). This specifically includes the clarification of the fermentation broth. Bleaching (Optional step (A.3)). This decolorization is preferably carried out in such a manner that the fermentation broth, from which microorganisms have been removed, is passed through a column with solid packing material in order to remove the dye by adsorption. Possible solid phases that can be used are, for example, diatomaceous earth or ion exchange packing material. This decolorization is preferably performed when the fermentation broth contains colored substances that may disrupt subsequent crystallization in step (B).

[0081] Crystallization and separation of aminobenzoic acid from fermentation broth containing aminobenzoate anions (steps (B) and (C)) In step (B) of this method, aminobenzoic acid is extracted from the fermentation broth containing aminobenzoate anions. precipitation (crystallization) This step is carried out in technical equipment suitable for crystallization, and is known in the field as... crystallizer .

[0082] Suitable crystallizers are, for example, stirred tanks or forced circulation crystallizers, such as the "Oslo type" crystallizer. In the crystallizer, the pH is adjusted to 3.0 to 5.5, preferably 3.5 to 4.5, more preferably 3.8 to 4.2, and most preferably 4.0. This is achieved by adding formic acid in its pure form (anhydrous) or preferably mixed with water. If a mixture of formic acid and water is used, the mass concentration of formic acid in the mixture is preferably at least 20%, more preferably 60% to 98%, and most preferably 70% to 95%, based on the total mass of the mixture. This adjustment of pH results in the formation of aminobenzoic acid anions (H₂NC₆H₄COO₃). - It is mainly or even completely converted into the electroneutrally neutral form (H2NC6H4COOH or H3N). + C6H4COO - ) and its crystals. This type of crystallization is also called reaction Crystallization. Then, in step (C), the crystallized aminobenzoic acid is removed; this can be done by known methods, such as, in particular, filtration, sedimentation, or centrifugation, leaving... Contains formic acid Mother liquor of root anions The mother liquor contains a dissolved amount of aminobenzoic acid.

[0083] Depending on the concentration present, removing a portion of the water may be useful to facilitate the separation of aminobenzoic acid in step (C) and / or prevent the volume of the mother liquor introduced into the fermentation from becoming excessive. Such a step can be performed before crystallization in step (B) (i.e., after step (A.2) and before step (B)) or after (i.e., after step (B) and before step (C), preferably before. Water can be removed by evaporation or membrane methods. All evaporation equipment known in the field is suitable for evaporation. To minimize heat load, evaporation is preferably carried out under reduced pressure, particularly at 0.1 mbar to 900 mbar, more preferably at 100 mbar to 500 mbar. This allows water to evaporate gently at temperatures from 45°C to 97°C, particularly up to 82°C, at which the decarboxylation of aminobenzoic acid, or, if evaporation is carried out after step (B), the decomposition of formic acid, can be virtually completely prevented.

[0084] It has been found useful to feed the fermentation broth and formic acid into the crystallizer via spatially separated feed devices (as far apart as possible). This achieves the effect of very good mixing of the reactants with the reactor contents before the acid-base reaction occurs. Examples of useful feed devices include pipes, preferably with isolation valves. In one embodiment, the feed devices for the fermentation broth and the acid are positioned opposite each other on the reactor wall, (substantially) at right angles. In another embodiment, the feed devices for the fermentation broth and the acid are arranged (substantially) parallel to the reactor wall, wherein the feed devices are opposite each other and as close as possible, especially adjacent to the reactor wall.

[0085] The crystallizer can be divided into chambers using suitable internal components. The flow direction can be adjusted by selecting the geometry and operating mode of the agitator. An external pump circulation system can also be provided for the crystallizer, in which case one of the two reactants—fermentation broth or formic acid—is introduced into the pump circulation system, and the other is introduced directly into the crystallizer. If the crystallizer is operated with a sieve and pump circulation system, the pump circulation system is located on the sieve base or side of the sieve used for fluidization.

[0086] Crystallization in the crystallizer can be carried out continuously or in batches. Continuous operation is superior. Regardless of the operating mode (continuous or batch), the exact operating parameters are determined (in particular) by the desired crystal size, which can be adjusted by residence time / reaction time and supersaturation level (longer residence time / reaction time and lower supersaturation level promote larger crystal size).

[0087] Crystallization is preferably carried out in the presence of seed crystals: In batch crystallization, the preferred procedure is to first add the fermentation broth to the crystallizer and adjust it to a specified temperature (preferably 5°C to 40°C, e.g., 20°C). If the pH of the fermentation broth is significantly higher than the solubility limit of aminobenzoic acid at the selected temperature, the fermentation broth is first slightly acidified with formic acid to a pH corresponding to or at least close to the solubility limit of aminobenzoic acid under the selected temperature and given boundary conditions (preferably pH 5.0 to 6.5). This slight acidification can be achieved rapidly. Then, seed crystals of the desired aminobenzoic acid polymorph are added (preferably polymorph (form) I in the case of o-aminobenzoic acid). This polymorph has relatively low solubility, thus facilitating a large recovery of aminobenzoic acid. The amount of seed crystals added is preferably about 0.1% to 1% of the aminobenzoic acid dissolved in the fermentation broth. In this way, a suspension of seed crystals is obtained (see also WO 2017 / 085170 A1). Subsequently, the pH is adjusted to 3.0 to 5.5, preferably 3.5 to 4.5, more preferably 3.8 to 4.2, and most preferably 4.0, by adding additional formic acid. The formic acid is preferably added gradually; for example, using 1 kg of the initially added fermentation broth and 75% formic acid over 1 hour. After the acid addition is complete, stirring continues for a certain period, particularly the same time as the time taken to add acid after the seed crystals. The precipitated aminobenzoic acid is then separated, particularly by filtration (under vacuum if necessary), sedimentation, or centrifugation (preferably by centrifugation), and preferably by washing more than once (especially twice) with a washing solution containing water, particularly acidic, and preferably formic acid, at a pH of 3.0 to 5.5, preferably 3.0 to 4.5, more preferably 3.2 to 3.8, and most preferably 3.5. Depending on the purity required for the intended subsequent application, the aminobenzoic acid can also be purified by recrystallization.

[0088] In continuous crystallization, seed crystals are typically added only at the start of the continuous process, as more seed crystals will subsequently form in situ (called secondary nucleation) or be present in sufficient quantities. The seed crystal suspension required for startup can be provided for batch crystallization as described above. Post-treatment (removal and washing of the crystals of aminobenzoic acid) can also be performed as described above.

[0089] The formic acid required for the precipitation of aminobenzoic acid can, in principle, come from any source, as long as it is not obtained by reduction of carbon dioxide, especially by chemical or electrochemical reduction, as further described below for preferred embodiments.

[0090] The mother liquor containing formate anions is introduced into the fermentation process (step (D)). Based on step (A.1) in step (D), the product generated in step (C) will be... Introduction of mother liquor containing formate anions FermentationThe aim is to select the largest possible proportion of the mother liquor introduced into the fermentation process in order to maximize the advantages of the invention; however, it may be necessary not to introduce a relatively small portion of the mother liquor into the fermentation process to avoid the accumulation of impurities (referred to as...) during the process. Purge A portion of the mother liquor is used to remove impurities. This removal can be performed periodically or intermittently, and when performed, it preferably includes 0.5% to 45.0% of the amount of mother liquor produced in step (C), more preferably 1.0% to 25%, and most preferably 1.5% to 5.0%. The remaining portion of the mother liquor produced in step (C), preferably 55.0% to 99.5%, more preferably 75.0% to 99.0%, and most preferably 95.0% to 98.5%, is then fed into the fermentation process according to step (A.1).

[0091] The mother liquor is discharged after depletion of aminobenzoic acid, followed by wastewater treatment. Depletion of aminobenzoic acid can be achieved by methods known in the art. Examples include extraction with an organic solvent and adsorption on an adsorbent, followed by desorption. The organic solvent used is preferably an alkanol having 8 to 12, more preferably 9 to 11 carbon atoms, particularly 1-decyl alcohol. Extraction is preferably performed as described on page 19, lines 15 to 33 of WO 2023 / 117756 A1. Dissolved aminobenzoic acid can be obtained by evaporation of the extractant or by back-extraction, as described on page 20, line 1 to page 21, line 8 of WO 2023 / 117756 A1. The adsorbent used is preferably activated carbon or a polymer adsorbent, particularly adsorbents based on polystyrene and / or polydivinylbenzene. Typical pore sizes are in the range of 1.5 to 65 nanometers, for example, 4.5 to 10 nanometers. Types of adsorbents with micropores and macropores are also available. For example, commercially available products can be found under the following brands: Lewatit (e.g., OC 1064 MD PH or AF 5), Macronet (e.g., MN 270, MN 202, MN 100 or MN 102), PuroSorb (e.g., PAD600), AmberSorb (e.g., L493 or 560), and Amberlite (e.g., XAD4). These adsorbents (as well as other adsorbents such as activated carbon) are suitable for acidic (as described in WO 2018 / 114841 A1) and alkaline (as described in WO 2015 / 124687 A1) desorption. If desorption is carried out under acidic conditions, it is recommended to perform additional regeneration of the adsorbent with alkali from time to time (e.g., after five acidic desorption cycles). In this way, the adsorption bed is free of organic impurities. Regardless of the nature of the desorption, the desorbate is preferably fed into the crystallizer according to step (B).

[0092] Regardless of its size, the portion of the mother liquor obtained in step (C) that is introduced into the fermentation according to step (A.1) is preferably premixed with nutrients, such as the aforementioned carbon and nitrogen sources or salts, such as potassium phosphate or calcium chloride, or trace elements, such as ferric sulfate, manganese sulfate, copper sulfate, zinc sulfate, or nickel chloride. Additionally, an antifoaming agent, such as polypropylene glycol, may be added during fermentation.

[0093] Before introducing the mother liquor containing formate anions into the fermentation according to step (A.1), the pH of the mother liquor can be raised to >5.5, particularly 5.6 to 11, by adding alkali (e.g., by adding ammonia water or gaseous ammonia). However, as already mentioned, surprisingly, this is not absolutely necessary for maintaining a constant pH during fermentation. Therefore, it is preferable to introduce the mother liquor containing formate anions into the fermentation according to step (A.1) without altering the pH value. Especially when pH adjustment is not required, to prevent the addition of formate from exceeding its degradation rate (and to prevent the pH from dropping too much as a result), the mother liquor containing formate anions should be gradually added to the fermentation according to step (A.1). Therefore, it is preferable to set the rate of mother liquor addition such that the pH of the fermentation broth does not change too much when the mother liquor is added.

[0094] The mother liquor containing formate anions can be introduced into the fermentation according to step (A.1) in such a way that the mother liquor is fed into the same fermentation reactor from which the aminobenzoic acid precipitated in step (C) (more precisely: the aminobenzoic acid anion is converted to aminobenzoic acid in step (B)) originally originated. Multiple fermentation reactors can also be operated in series, with the resulting mother liquor fed into downstream fermentation reactors. To start the process, when the mother liquor containing formate anions is not yet available, the pH of the fermentation can be controlled by adding an alkali (e.g., by adding ammonia or gaseous ammonia, an aqueous solution of potassium hydroxide, or an aqueous solution of sodium hydroxide). Preferably, the pH is controlled by adding an aqueous solution of potassium hydroxide or sodium hydroxide during startup.

[0095] In the case of a discontinuous process, the procedure can also involve feeding the mother liquor containing formate anions obtained in one fermentation cycle into the downstream fermentation process. cycle In this context, it is irrelevant whether all fermentation cycles are carried out in the same fermentation reactor or in different fermentation reactors. In the first fermentation cycle, of course, no mother liquor is available. This first fermentation reactor is then operated under pH control by adding alkali (e.g., by adding ammonia water or gaseous ammonia, potassium hydroxide aqueous solution, or sodium hydroxide aqueous solution).

[0096] As previously mentioned, it was unexpected that introducing a mother liquor containing formate anions into fermentation could be achieved without significant formate anion accumulation in the fermentation broth. Instead, the formate anions are oxidized to carbon dioxide, bound to the fermentation target product aminobenzoate anion, and / or metabolized to build biomass. If desired, formate degradation during fermentation can be increased by adding formate-degrading enzymes. Examples of suitable enzymes are formate oxidase (EC 1.2.3.1) and formate dehydrogenase (EC 1.17.1).

[0097] Aminobenzoic acid is used to produce other valuable products. After any necessary further purification using known methods (e.g., recrystallization), the aminobenzoic acid obtained in step (C) is suitable for all applications of aminobenzoic acid known in the art. For example, anthranilic acid is an important raw material for the synthesis of anthranilic esters, which are important flavor enhancers, indigo additives, and pharmaceutical and crop protectants (acaricides). The anthranilic acid produced according to the present invention is suitable for all these purposes.

[0098] The production of polymers preferably uses aminobenzoic acid produced according to the present invention. Hereinafter, the method according to the present invention can make a valuable contribution to the more sustainable production of plastics, which are typically in high demand. For example, anthranilic acid can be converted into poly(anthranilamide), as described in WO 2022 / 008449 A1 (after conversion to indocyanine anhydride) or WO 2022 / 008450 A1 (after conversion to anthranilic ester). As described in WO 2022 / 122906 A1, the anthranilic acid obtained according to the present invention can also be converted into anthranilic acid derivatives selected from anthranilic benzoyl halides, indocyanine anhydride, or mixtures thereof, and then reacted with a polyol to form a polyamine, which is then suitable for phosgenation to form the corresponding polyisocyanate.

[0099] In particular, the aminobenzoic acid obtained according to the present invention can also be decarboxylated to aniline, an important raw material, especially in the polyurethane industry. As further described below, using the aminobenzoic acid produced according to the present invention for the production of aniline provides the possibility of utilizing the synergistic effect with the production of aminobenzoic acid through fermentation and crystallization, and is therefore particularly preferred.

[0100] Aniline obtained in this manner can then be used for all known applications. In particular, these include (acid-catalyzed) reactions with formaldehyde to form methylene diphenylene diamine and polymethylene polyphenylene polyamine, which are phosgenated to form methylene diphenylene diisocyanate and / or polymethylene polyphenylene polyisocyanate, and subsequent reactions with polyols to form polyurethanes. Needless to say, aniline obtained by decarboxylation of aminobenzoic acid produced according to the invention can also be used for various applications, such as the production of azo compounds. For example, this includes the production of methyl red, which is obtained by diazotizing the amino group of o-aminobenzoic acid with sodium nitrite and hydrochloric acid, followed by azo coupling with N,N-dimethylaniline.

[0101] Formic acid is produced by decarboxylating aminobenzoic acid and using the resulting carbon dioxide. As already mentioned, decarboxylating aminobenzoic acid to aniline is a particularly preferred further configuration of this method. The released carbon dioxide can be converted into formic acid by methods known in the art (particularly chemical or electrochemical reduction). Formic acid It can also be used in step (B). This means that, aside from potential losses, the amount of formic acid available is the same as the stoichiometric amount required to convert the aminobenzoate anion to aminobenzoic acid. This is because, according to stoichiometry... • One mole of aminobenzoic acid is required to produce one mole of aminobenzoic acid from the aminobenzoate anion. • In the process of decarboxylation to aniline, every mole of aminobenzoic acid reacts to produce 1 mole of carbon dioxide. • It takes 1 mole of carbon dioxide to reduce and produce 1 mole of formic acid.

[0102] Therefore, this implementation scheme makes it possible to provide the stoichiometric amount of total formic acid required by the recycling scheme under ideal conditions (aside from, for example, yield losses). Furthermore, the stoichiometric amount of carbon dioxide released during decarboxylation is re-chemically bonded (and not released into the atmosphere), which is advantageous in itself.

[0103] Decarboxylation Decarboxylation can be carried out in principle as is known in the prior art. A catalyst can be used, but it is not mandatory.

[0104] Examples of suitable catalysts are aqueous acids, such as sulfuric acid, nitric acid, and hydrochloric acid; solid acids such as zeolites and Si-Ti molecular sieves; solid bases such as hydroxyapatite and hydrotalcite; and polymeric acids, such as ion exchange resins (preferably Amberlyst). Particularly preferred catalysts are described in WO 2022 / 253890 A1. The catalysts disclosed therein are characterized by a high mass proportion of alumina (at least 40%). The alumina is preferably γ-Al₂O₃ or η-Al₂O₃, especially when no other metal oxides are present besides alumina. In principle, other metal oxides may also be present besides alumina, particularly containing magnesium oxide (MgO), in a mass proportion of 1.0% to 60.0%, preferably 2.0% to 50.0%, particularly preferably 5.0% to 35.0%, based on the total mass of the metal oxides. Furthermore, the catalyst may contain SiO₂ in a mass proportion of 1.0% to 30.0%, preferably 2.0% to 20.0%, more preferably 2.0% to 10.0% based on its total mass.

[0105] Regarding reaction conditions, decarboxylation can be carried out over a wide range of temperatures and pressures. A suitable reaction temperature is preferably 150°C to 300°C, more preferably 160°C to 280°C, and most preferably 180°C to 240°C. The (absolute) reaction pressure can be 0.05 bar to 300 bar, preferably 1.0 bar to 100 bar, and more preferably 1.0 bar to 60 bar.

[0106] Decarboxylation can also be carried out in the presence of aniline, i.e., aminobenzoic acid dissolved in aniline. Since aniline catalyzes the decarboxylation of aminobenzoic acid, thereby accelerating its formation (autocatalysis), an external catalyst is not absolutely necessary in this variant; see also WO 2020 / 020919 A1, which describes this method. Unlike the procedure disclosed in WO2020 / 020919 A1, purified aniline can also be used as a solvent for aminobenzoic acid. Furthermore, unlike the procedure disclosed in WO2020 / 020919 A1, an external catalyst can be additionally used. In the case of a batch reaction, based on the total mass of aniline and aminobenzoic acid, it is preferable to decarboxylate... Before starting A mass ratio of aniline is established from 0.1% to 90%, preferably 1.0% to 70%, and particularly preferably 5.0% to 50%. In the case of a continuous reaction, based on the total mass of aniline and aminobenzoic acid, it is preferred to occur during the decarboxylation process. Constantly The mass ratio of aniline is set at 0.1% to 90%, preferably 1.0% to 70%, and particularly preferably 5.0% to 50%.

[0107] Besides aniline, other solvents or diluents, especially water, can of course be used. Further suitable are organic, polar or protic solvents, such as halogenated aliphatic or aromatic hydrocarbons, linear or cyclic ethers, linear or cyclic esters, linear or cyclic amides, alcohols, ketones, nitriles, phenol derivatives, benzoylaniline, sulfonamides or sulfolane, preferably having a boiling point above a selected reaction temperature under selected conditions, and preferably forming a homogeneous reaction mixture with the reaction components at that temperature.

[0108] Both gas-phase and liquid-phase reactions are suitable for the reaction scheme. The reaction can be carried out continuously (preferred) or in batches.

[0109] The preferred procedure includes decarboxylating aminobenzoic acid. • In the liquid or gas phase of a reactor, especially in a tubular reactor, there is an integrated catalyst fixed bed (comprising a catalyst bed in the form of a shaped body (extrusion) or a catalyst in the form of an integral structure). • In the liquid phase or preferably gas phase of a fluidized bed reactor, or • In the liquid phase of a stirred tank, there is a suspension containing the catalyst ( slurry ).

[0110] In this article, tubular reactor This is understood to refer to a tubular reactor, through which the reaction mixture flows during operation in a continuous reaction scheme (which is preferred). Tubular reactors with a small length-to-diameter ratio are also called... Tower-type counter Applicator As understood herein, they are also included in the term "tubular reactor," just as tubular reactors, such as bubble column reactors, are specific configurations.

[0111] The use of shaped catalysts (extrusions) or monolithic catalyst structures allows for easy reuse of the catalyst after decarboxylation.

[0112] The catalyst remaining after decarboxylation is preferably regenerated before reuse. To this end, the catalyst can be washed with an organic solvent or aqueous solution and / or burned off at high temperature in the presence of O2 to remove organic deposits.

[0113] Using standard techniques, particularly distillation, the formed aniline can be separated and purified, and used as described above.

[0114] Chemical or electrochemical reduction of carbon dioxide As mentioned above, the carbon dioxide produced during decarboxylation is preferably converted into formic acid. For this purpose, carbon dioxide from decarboxylation is particularly suitable because the impurities present therein are at most trace amounts of aniline or anthranilic acid, which can be easily removed.

[0115] Another system-inherent source of carbon dioxide that can be used for the conversion to formic acid is the carbon dioxide produced during fermentation according to step (A.1) (particularly through the oxidation of the formate anion). However, this source of carbon dioxide is less preferred because the carbon dioxide thus obtained is produced in a diluted form. Needless to say, carbon dioxide from any external source can also be used, provided that it is produced with sufficient purity or can be purified with acceptable effort.

[0116] The conversion of carbon dioxide to formic acid is achieved by methods known in the prior art, and will therefore be only briefly reported below.

[0117] The chemical reduction of carbon dioxide is achieved by hydrogenation with hydrogen using suitable catalysts, particularly ruthenium and iridium catalysts with nitrogen and / or phosphorus ligands. Suitable methods are described, for example, by W.-H. Wang, Y. Himeda, J.M. Buckerman, GF. Manbeck, and E. Fujita. Chem. Rev. 2015, 115 , 12936-12973 (“ CO 2 Hydrogenation to Formate and Methanol as an Alternative to Photo- and Electrochemical CO 2 Reduction In [1], for the current relevant purpose of using formic acid in the crystallization of aminobenzoic acid, the method of choice should be one in which the final product is actually formic acid (as described in scheme 2 of [1]) rather than formate. (In principle, formate can certainly be readily converted to formic acid; however, this additional step will have a considerable impact on the economic feasibility of the method.) T. Schaub and R.A. Paciello in Angew. Chem. 2011, 123 , 7416 –7420 (“ Ein Verfahren zur Herstellung von Ameisensäure durch CO 2 -Hydrierung: Thermodynamik und die Rolle von CO [3] describes a method using ruthenium catalysts, particularly [Ru(H)2(P] n The method described in Bu3)4 uses amines, particularly trihexylamine, and polar solvents (capable of forming hydrogen bonds) such as glycols (particularly 2-methyl-1,3-propanediol, 1,3-propanediol, 1,2-propanediol, or ethylene glycol). The disclosed method is a multiphase liquid-liquid process concept that allows for the recovery of amines, polar solvents, and catalysts, and the removal of formic acid from the system by distillation.

[0118] The electrochemical reduction of carbon dioxide is achieved through acidic electrolysis, specifically at a concentration below the pK of formic acid.a (3.77) at pH, particularly at pH 2.00 to 3.75. M. Oßkopp, A. Löwe, CMS Lobo, S. Baranyai, T. Khoza, M. Auinger and E. Klemm in Journal of CO 2 Utilization 2022, 56 , 101823(“ Producing formic acid at low pH values ​​by electrochemical CO 2 reduction The electrolyzer and operating mode suitable for this purpose are described in "”[5].

[0119] The above method makes it possible to reduce salt pollution in wastewater and increase the yield of aminobenzoic acid because at least most of the mother liquor is recycled back into the fermentation process, meaning that no aminobenzoic acid dissolved therein is lost. This method will be illustrated by the following examples.

[0120] Example: Example 1 (Precipitation of aminobenzoic acid with formic acid) The fermentation broth, initially pH 7.8, containing 55.9 g / L (calculated as aminobenzoic acid) of aminobenzoate anions, was adjusted to pH 5.7 within 30 minutes by adding formic acid (≥98%). Subsequently, aminobenzoic acid seed crystals were added in an amount equivalent to 1% of the calculated mass of aminobenzoic acid present in the fermentation broth. After waiting 30 minutes, the pH was lowered to pH 3.7 by adding additional formic acid over 60 minutes. The mixture was continuously stirred at 200 rpm. The resulting suspension of aminobenzoic acid in the mother liquor was filtered through a suction filter under vacuum using a diaphragm pump. The moist filter cake was washed with aqueous hydrochloric acid solution (pH 3.5) and dried overnight at 60°C and 30 mbar. The yield of isolated and dried aminobenzoic acid was 79% by weight.

[0121] Example 2 (Degradation of formate by Corynebacterium glutamicum) By inoculating Corynebacterium glutamicum ATCC 13032 into Erlenmeyer flasks OD 600 Cells were prepared by incubating in 0.005% brain-heart infusion medium (out-of-the-box formulation from Oxoid) at 30°C and 200 rpm for 24 hours. Cells were then harvested by centrifugation at 8000 rpm and 4°C for 10 minutes. The cell pellet was stored at -20°C until use.

[0122] Thaw the cell pellet and then resuspend it in McIlvaine buffer (citrate phosphate buffer, such as "ABUFFER SOLUTION FOR COLORIMETRIC COMPARISON", TC McIlvaine). Journal of Biological Chemistry , 1921, 49 (1), 183–186 (doi:10.1016 / S0021-9258(18)86000-8) [4]) prepared as described. In 2 mL reaction volumes of overnight culture tubes, in McIlvaine buffer at different pH values, at 5.0 normalized OD 600 The reaction was initiated by adding sodium formate after formate degradation. The tubes were incubated at 30°C and 200 rpm. Samples for formate quantification were collected at 0, 2, 4, 6, and 24 hours and incubated at 95°C for 5 minutes to inactivate the formate. Cellular components were then removed by centrifugation at 13000 rpm for 5 minutes. The supernatant was stored at 4°C until formate quantification. Formate was photometrically quantified by enzymatic formation of NADH. The sample was diluted in reaction buffer (50 mM potassium phosphate buffer, pH 7.6) according to the expected formate concentration. 1 U / mL of Candida botrytis cinerea (…) was added. C. boidinii Formate dehydrogenase and 1 mM NAD + The reactants were then incubated at 37°C for 3 hours. Afterward, the absorbance was measured at 340 nm, and the formate concentration was determined using a calibration curve.

[0123] Table 1 shows the changes in formate concentration over time. The fastest degradation of formate was observed at pH 6.0.

[0124] Table 1: Changes in formate concentration over time at different pH values. mM = mmol / L Example 3 (Fermentation of Corynebacterium glutamicum using a mother liquor containing formate) Adding a stock solution containing sodium formate to a Corynebacterium glutamicum culture The microbial strains used are as follows: Starting with Corynebacterium glutamicum ATCC13032, a microbial strain for the production of anthranilic acid was generated through targeted chromosome modification. All gene modifications, namely chromosome deletions and gene integrations, were performed using the corresponding pK19... mobsacB This is achieved through dual homologous recombination of derivatives (Schäfer et al., 1994: " Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. ” Gene 145(1):69-73. doi: 10.1016 / 0378-1119(94)90324-7) [6].

[0125] The activity of anthranilate phosphoribosyltransferase TrpD is mediated by the initial loss of the native protein. trpD Allele (SEQ ID NO. 1) and alleles with a GTG start codon instead of an ATG start codon and a ribosome binding site with a reduced distance from the start codon (referred to as...) trpD5 The reduction is achieved by replacing (SEQ ID NO. 2).

[0126] In addition, phosphoenolpyruvate carboxylase is inactivated by the deletion of most of the corresponding gene, as described in (SEQ ID NO.3).

[0127] To enhance the biosynthetic pathway of aromatic compounds, under the control of the constitutive promoter of the elongation factor Tuf, a kinase encoding shikimate from Escherichia coli is activated. aroL ( b0388 The gene encodes shikimate-1-carboxylvinyltransferase 3-phosphate from Corynebacterium glutamicum. aroA ( cg0873 The gene and encoding the branching acid synthase from Corynebacterium glutamicum. aroC ( cg1829 Artificial polycistronic P genes tuf - aroLAC The operon (SEQ ID NO. 4) is integrated into cg2563 Downstream. Furthermore, the body P will be constructed. tuf - aroG new (SEQ ID NO. 5) integrated into cg3132 In the genome of downstream strains, this construct is present in P tuf The promoter encodes a feedback resistance variant of DAHP synthase from Escherichia coli.

[0128] Corynebacterium glutamicum strains were pre-cultured by transferring them from a low-temperature culture to sterile SY medium (10 g / L yeast extract, 16 g / L soybean peptone, 5 g / L NaCl, 16 g / L glucose), and then incubated at 30°C and 200 rpm in 1 L Erlenmeyer flasks containing 50 mL of liquid culture until OD was reached. 600Approximately 30. Transfer 5 mL / vial of this first preculture to sterile modified CGXII medium (5 g / L yeast extract, 10 g / L (NH4)2SO4, 1 g / L KH2PO4, 1 g / L K2HPO4, 0.25 g / L MgSO4 x 7 H2O, 0.01 g / L CaCl2 x 2 H2O, 5 g / L urea, 63 g / L 3-(N-morpholino)propanesulfonic acid (MOPS), 10 mg / L MnSO4 x H2O, 10 mg / L FeSO4 x 7 H2O, 1 mg / L ZnSO4 x 7 H2O, 0.2 mg / L CuSO4 x 5 H2O, 0.02 mg / L NiCl2 x 6 H2O, 2 mg / L biotin, 40 g / L glucose) and incubate under the same conditions until OD. 600Approximately 40. As with the master culture, the final growth stage (“seed”) was carried out under aerobic conditions (dissolved oxygen concentration ≥30%) in a stirred bioreactor (BioBlock, Eppendorf). 80 mL of the second preculture was transferred to 520 mL of seed culture medium (8.33 g / L yeast extract, 5 g / L (NH4)2SO4, 6.67 g / L KH2PO4, 6.67 g / L K2HPO4, 3.33 g / L MgSO4 x 7 H2O, 0.07 g / L CaCl2 x 2H2O, 4.17 g / L PPG 2000, 167 mg / L MnSO4 x H2O, 167 mg / L FeSO4 x 7 H2O, 16.7 mg / L ZnSO4 x 7 H2O, 3.34 mg / L CuSO4 x 5 H2O, 0.334 mg / L NiCl2 x 6 H2O, 3 mg / L biotin, 50 g / L granulated corn steep liquor, 33 g / L glucose). Seed fermentation was carried out at 30°C and pH 7.0. The feed consisted of a glucose-tryptophan mixture (480 g / L glucose, 1.6 g / L tryptophan) and a mixture of NaOH and NH3 solution (1.55 mol / L NaOH, 8% NH3 by mass) for pH control. Once the OD reaches approximately 200, in each case, 80 mL of seed culture is transferred to each master culture batch containing 320 mL of medium (5 g / L (NH4)2SO4, 10 g / L KH2PO4, 10 g / L K2HPO4, 5 g / L MgSO4 x 7 H2O, 0.1 g / L CaCl2 x 2 H2O, 1.25 g / L PPG 2000, 125 mg / L MnSO4 x H2O, 125 mg / L FeSO4 x 7 H2O, 12.5 mg / L ZnSO4 x 7 H2O, 2.5 mg / L CuSO4 x 5 H2O, 0.25 mg / L NiCl2 x 6 H2O, 5 mg / L biotin, 50 g / L glucose). The primary fermentation was carried out at 33°C and pH 7.0, with a feed of 600 g / L glucose solution, a 20% NH3 solution for pH control, and a mother liquor or synthetic solution containing sodium formate from Example 1.

[0129] Before use, the stock solution was adjusted to pH 7.0 with NaOH solution and aseptically filtered. The formate concentration was 42.3 g / L (determined using a Cedex Bio HT analyzer, Roche Custom Biotech); the anthranilic acid concentration was 9 g / L (determined using a 1260II Infinity HPLC system, Agilent Technologies).

[0130] As a substitute for the actual mother liquor, a synthetic solution containing 35.7 g / L sodium formate (24.2 g / L formic acid) and 8 g / L anthranilic acid was prepared, adjusted to pH 7.0 with NaOH solution, and aseptically filtered. Different addition rates were selected in cultures containing the synthetic solution. For simplicity, they are designated as "low" and "high" in Tables 2 through 6. Adding formate at the "high" addition rate of the synthetic solution corresponds to the addition rate in the metered addition of the mother liquor. Adding formate at the "low" addition rate of the synthetic solution is 20% lower.

[0131] like Table 2 As shown, no culture exhibited a sharp increase in formate concentration. Regardless of the addition rate and differences in formate concentration in the feed solution, >99% of the feed formate was degraded by the microorganisms in each case. Table 3 As shown, this allows for the production of anthranilic acid from feedstock (synthetic or actual) mother liquor at approximately 48% to 139% Na, depending on the batch. + Counterions. Anthranilic acid recovered from the mother liquor is not considered in the production of anthranilic acid, as it has already been neutralized during mother liquor preparation. Depending on the batch, the recovered anthranilic acid accounts for approximately 5.4% to 9.8% of the final anthranilic acid.

[0132] Table 2: Maximum formate concentration and proportion of formate consumed [a] Under the same conditions, two tests were performed in different reaction apparatuses; the two tests performed in this manner are designated below as “(1)” and “(2)”.

[0133] Table 3: The ratio of sodium counterions in the mother liquor feed to the generated anthranilic acid is determined by the synthesis process. Tables 4-6 show the absolute amounts of dry microbial biomass, anthranilic acid, and degraded formate at different culture periods. This batch had slightly lower amounts of dry biomass and anthranilic acid due to temporary glucose restriction in the "mother liquor (1)" batch after approximately 20 hours. However, the fact that the amount of degraded formate shown in Table 6 is higher than that in the "mother liquor (2)" indicates that the cells' ability to metabolize formate was not limited. In conclusion, the results suggest that growth and product formation are possible with the simultaneous recovery of the formate-containing mother liquor.

[0134] Table 4: During the cultivation process using a mother liquor / solution containing formate as feed, the absolute amount of dry microbial biomass Table 5: The absolute amount of anthranilic acid during the culture process using a mother liquor / solution containing formate as feed. Table 6: The absolute amount of formate degraded during the culture process of a mother liquor / solution containing formate as feed. Example 4 (Degradation of formate by different microorganisms) Cultivation of different microorganisms for formate degradation Table 7 summarizes the culture conditions. Pre-cultures were inoculated with cell material from cryogenic or solid media and incubated overnight. Master cultures were inoculated at a ratio of 1 / 200 and cultured for 48 hours, followed by centrifugation to collect cells.

[0135] Table 7: Cultivation conditions Nutrient broth culture medium For 1 liter of liquid culture medium, dissolve 25 g of ready-to-use powder (Invitrogen / Thermo Fisher Scientific) in 1 liter of deionized water. The liquid culture medium contains: •3 g / L yeast extract •1 g / L glucose •6 g / L NaCl • 15 g / L peptone Autoclave the solution and then store it at room temperature.

[0136] Yeast malt culture medium For 1 liter of liquid culture medium, dissolve 21 g of ready-to-use powder (NutriSelect) in 1 liter of deionized water. The liquid culture medium contains: •10 g / L glucose •3 g / L malt extract •5 g / L peptone •3 g / L yeast extract Autoclave the solution and then store it at room temperature.

[0137] Yeast peptone glucose (YPD) medium For 1 L of liquid culture medium, prepare a 100 g / L (5x) glucose monohydrate solution (solution 1). As a second solution, dissolve 20 g peptone and 10 g yeast extract in deionized water and bring the volume to 0.8 L. Autoclave each solution separately and then store at room temperature. For the final culture medium, add 0.8 L of solution 2 to 0.2 L of solution 1. The liquid culture medium contains: • 20 g / L glucose monohydrate • 20 g / L peptone • 10 g / L yeast extract Pichia pastoris was cultured using a modified YPD medium. The carbon source used was 0.5% (v / v) methanol instead of glucose monohydrate. Additionally, a 10% (v / v) 100 g / L (NH₄)₂SO₄ solution and a 0.2% (v / v) 200 mg / L biotin solution were used. These solutions were prepared with deionized water and aseptically filtered. The biotin solution was stored at 4°C, while the (NH₄)₂SO₄ solution was stored at room temperature. To obtain the same concentrations as in the original YPD medium, 9.3% (v / v) deionized water and autoclaved water were added.

[0138] Formate degradation reaction Formate degradation was carried out in 15 mL culture tubes containing 2 mL of reaction solution in McIlvaine buffer at 20 °C and 200 rpm. pH 5 was used for yeast and pH 6 for bacteria. Cells were used for final OD. 600 In a reaction with a concentration of 5, equal volumes of cell culture were centrifuged at 9000 rpm for 10 minutes after culturing, and the cell pellet was then resuspended in reaction buffer. The reaction was initiated by adding 7.5 mM sodium formate. During the reaction, samples were collected for formate quantification.

[0139] Quantitative determination of formate using MTP-based FDH spectrophotometry Formate in samples was quantified using a robust FDH assay based on formate dehydrogenase CbFDH. Table 8 lists the required solutions, including volume and concentration. Sodium formate solution or the sample was used in the case of a standard curve. A dilution for the standard curve assay was prepared using potassium phosphate buffer. (From NAD...)+ A master mixture corresponding to the volume required for the assay was prepared in a solution of CbFDH and buffer, and pipetted into each well to begin the assay. Initially, either the sample or formate solution was added to the well. The assay was performed in a photometer at 37°C for 3 hours. Measurements were taken at 340 nm.

[0140] Table 8: The solutions, volumes, and concentrations used in the CbFDH determination, V tot = 200 µL All strains degraded significant amounts of formate. Although formate was no longer detectable in *Pichia pastoris* after 4.1 hours, 1.4 mM was still detectable in *Pseudomonas putida* and 6.2 mM in *Hansenula polymorpha*. After 24.5 hours, formate was no longer detectable in any strain.

Claims

1. A method for producing aminobenzoic acid, the method comprising the following steps: (A) Provides a fermentation broth containing aminobenzoate anions, comprising: (A.1) fermenting fermentable carbon-containing and nitrogen-containing compounds at a pH greater than 5.5 in the presence of microorganisms selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans, or a mixture of two or more of the above microorganisms; and (A.2) removing the microorganisms. (B) The pH of the fermentation broth containing aminobenzoate anions was set to 3.0 to 5.5 by adding formic acid to precipitate aminobenzoic acid; (C) Remove the aminobenzoic acid precipitated in step (B) to obtain a mother liquor containing formate anions and formic acid; and (D) Introduce 55.0% to 100% of the total mother liquor produced in step (C) into the fermentation according to step (A.1) and deplete the formate anions and formic acid by means of (i) Added enzyme preparations, and / or (ii) Microorganisms used in step (A) and / or (iii) Adding additional microorganisms that are different from the microorganisms used in step (A).

2. The method according to claim 1, wherein water is removed after step (A.2) and before step (B) or after step (B) and before step (C).

3. The method according to claim 1 or 2, wherein the nitrogen-containing compound is selected from ammonia, ammonia water, ammonium salt, soybean protein, urea, or a mixture of two or more of the above nitrogen-containing compounds.

4. The method according to any one of the preceding claims, wherein the fermentable carbonaceous compound is selected from starch hydrolysate, alkali metal formate or ammonium formate, sugarcane juice, beet juice, hydrolysate of lignocellulose raw material, or a mixture of two or more of the above carbonaceous compounds.

5. The method according to any one of the preceding claims, wherein a pH of 5.6 to 11 is observed in step (A.1).

6. The method according to any one of the preceding claims, wherein formic acid is used in the form of a mixture of formic acid and water or anhydrous formic acid, wherein the mass concentration of formic acid in the mixture is at least 20% based on the total mass of the mixture.

7. The method according to any one of the preceding claims, wherein anthranilic acid or para-aminobenzoic acid is produced.

8. The method according to any one of the preceding claims, wherein the aminobenzoic acid from step (C) is converted into aniline and carbon dioxide is eliminated.

9. The method of claim 8, wherein the carbon dioxide formed during the conversion to aniline is converted to formic acid, and the formic acid thus obtained is used in step (B).

10. The method according to any one of the preceding claims, wherein carbon dioxide is formed in the fermentation according to step (A.1) and converted into formic acid, and the formic acid thus obtained is used in step (B).

11. The method according to claim 9 or 10, wherein the conversion to formic acid is achieved by hydrogenation of carbon dioxide or acidic electrolysis.

12. The method according to any one of the preceding claims, wherein, In step (D), the first portion of the mother liquor is mixed with additional carbonaceous compounds, additional nitrogenous compounds, compounds for reducing foaming during fermentation, inorganic salts, trace elements, or a mixture of two or more of the above compounds before being introduced into fermentation.

13. The method according to any one of the preceding claims, wherein in step (D), less than 100% of the total mother liquor generated in step (C) is introduced into the fermentation according to step (A.1), the portion of the mother liquor not introduced into the fermentation is depleted of aminobenzoic acid, and then wastewater is treated.

14. The method according to any one of the preceding claims, wherein the enzyme preparation comprises formate dehydrogenase and / or formate oxidase.

15. The method according to claim 14, wherein formate dehydrogenase belongs to EC 1.17.1.9 and formate oxidase belongs to EC 1.2.3.1.

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