Production of aminobenzoic acid

Abstract

The invention relates to the production of aminobenzoic acid using a method having the steps of (A) providing an aminobenzoate anion-containing fermentation broth by carrying out a fermentation process in the presence of microorganisms selected from 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 a pH value of 3.0 to 5.5 in the aminobenzoate anion-containing fermentation broth by adding formic acid, (C) separating aminobenzoic acid precipitated in step (B), and (D) partly or completely introducing the mother liquor precipitated in step (C) to the fermentation, thereby depleting the formate anions contained in the mother liquor and the formic acid by means of (i) an added enzyme preparation and / or (ii) the microorganisms used in step (A) and / or (iii) added additional microorganisms which differ from the microorganisms used in step (A). The aminobenzoic acid obtained in this manner can be converted into secondary products, in particular into aniline and, in the case of ortho-aminobenzoic acid, into isatoic anhydride or anthranilic acid ester, which can be used as starting materials in the production of polymeric compounds such as polyurethanes or poly(anthranilamide).

Description

[0001] PRODUCTION OF AMINOBENZOIC ACID

[0002] The work leading to the present invention was financially supported by the German Federal Ministry of Food and Agriculture under grant number 2221NR073X.

[0003] The present invention relates to the production of aminobenzoic acid by a process comprising the steps of (A) providing a fermentation broth containing aminobenzoate anions by fermentation in the presence of microorganisms selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans or a mixture of two or more of the aforementioned microorganisms and subsequent clarification, (B) adjusting a pH in the fermentation broth containing aminobenzoate anions of 3.0 to 5.5 by adding formic acid, (C) separating aminobenzoic acid precipitated in step (B) and (D) partially to completely introducing the mother liquor obtained in step (C) into the fermentation with depletion of the formate anions and formic acid contained in the mother liquor by (i) an added enzyme preparation and / or (ii) the Microorganisms and / or (iii) added further microorganisms,which are different from the microorganisms used in step (A). The aminobenzoic acid thus obtained can be converted into secondary products, in particular aniline and, in the case of ortho-aminobenzoic acid, isatoic anhydride or anthranilic acid esters, which in turn can serve as starting materials for the production of polymeric compounds such as polyurethanes or poly(anthranilamide).

[0004] The production of organic acids through fermentation processes has received particular attention in recent times. Among the organic acids accessible by fermentation, aminobenzoic acid is an economically important product. Aminobenzoic acid is used, for example, in the production of dyes, fragrances, pesticides, and pharmaceuticals. Another example of an application of aminobenzoic acid is its use in the production of aniline by decarboxylation. Aniline, in turn, is particularly important as an intermediate in the production of isocyanates. The ortho isomer of aminobenzoic acid, anthranilic acid, can also serve as a starting material for the production of poly(anthranilamines) and polyamines and the corresponding polyisocyanates.

[0005] The fermentative production of aminobenzoic acid is generally known in the art; see, for example, international patent application WO 2015 / 124687 A1 (which describes the two-step production of aniline via ortho-aminobenzoic acid as an intermediate) and the literature cited therein. Fermentation processes take place in an aqueous environment and, in the case of the production of aminobenzoic acid, generally yield aqueous product mixtures (fermentation broths) with a mass content of aminobenzoic acid, particularly in the range of 10.0 g / L to 100 g / L.

[0006] The ortho isomer of aminobenzoic acid (anthranilic acid) is of particular importance. In the metabolism of bacteria and yeasts, anthranilic acid is formed in the shikimic acid pathway as a natural intermediate in the synthesis of tryptophan. In the biotechnological production of anthranilic acid, its conversion in the pathway is reduced or prevented to achieve accumulation in the fermentation medium. Such a concept for the biotechnological production of anthranilic acid and its subsequent catalytic conversion to aniline is described in the aforementioned international patent applications WO 2015 / 124686 A1 and WO 2015 / 124687 A1. The use of bacteria from the Corynebacteria or Pseudomonads families is described as a possible recombinant microorganism. A more recent application (WO 2017 / 102853 A1) describes the use of yeast.

[0007] Para-aminobenzoic acid is also of interest. The synthesis of para-aminobenzoic acid can occur in the metabolism of bacteria and yeasts via the intermediate chorismate, which is formed as an intermediate in the shikimic acid pathway. Chorismate is first enzymatically converted to 4-amino-4-deoxychorismate and then, through a second enzymatic reaction, to para-aminobenzoic acid. A concept for the biotechnological production of aniline via the intermediate para-aminobenzoic acid is described in the international application US 2016 / 068876 A1. The use of bacteria from the Corynebacteria family is also described here as a possible recombinant microorganism.

[0008] The fermentative production of aminobenzoic acid produces an aqueous product stream. When fermentation is carried out at pH values ​​significantly above the isoelectric point (the usual pH range when using bacteria as microorganisms), the valuable product, aminobenzoic acid, is present predominantly to fully charged as an anion (aminobenzoate anion). To neutralize the aminobenzoic acid formed during fermentation, a base, particularly sodium hydroxide, is typically added as a pH adjuster. After carrying out the workup steps customary for fermentation, the valuable product, aminobenzoic acid, can be precipitated in an electroneutral form as a solid (crystallization) by adjusting the pH to a value near or at the isoelectric point. The aminobenzoic acid can then be separated, for example, by filtration.The filtered product initially occurs in a highly water-containing state ("slurry"). This product is then washed, for example, and dried if necessary (depending on the intended use), or dissolved in a solvent such as aniline or 1-dodecanol (see WO 2015 / 124687 A1). Mineral acids, especially hydrochloric acid, are typically used to adjust the pH during crystallization.This is described, for example, in the international application WO 2017 / 085170 A1, which deals with a process for producing aminobenzoic acid or an aminobenzoic acid derivative by fermenting a suitable raw material under the influence of suitable microorganisms to obtain a fermentation broth comprising aminobenzoate and / or aminobenzoic acid. The focus is particularly on obtaining the aminobenzoic acid from the fermentation broth by crystallization using a one-step acid treatment in the presence of seed crystals. However, the use of mineral acids in the crystallization process is inevitably associated with the formation of the corresponding salts, particularly sodium chloride. This salt load represents a significant wastewater burden and is therefore disadvantageous from a commercial and economic perspective.

[0009] As an alternative to the use of mineral acids, in a specific embodiment of the fermentative production of aminobenzoic acid, carbon dioxide can also be introduced under pressure to crystallize the aminobenzoic acid (crystallization with "carbonic acid"). Such a process is described in WO 2019 / 234092 A1 (also published as US 2021 / 0222215 A1). In this process, the fermentation takes place in the presence of a calcium salt, wherein (I) the aminobenzoic acid present in the fermentation broth is partially bound, optionally as far as possible due to the solubility equilibrium, as insoluble calcium aminobenzoate,This insoluble calcium aminobenzoate is then (II) isolated either as such or in admixture with the microorganisms used in the fermentation and converted into a water-soluble form by separating an insoluble calcium salt different from calcium aminobenzoate [ion exchange], and then (III) aminobenzoic acid is precipitated by introducing carbon dioxide under pressure into the aqueous solution freed from the precipitated calcium salt [crystallization]. The advantages of this process are a reduction in the concentration of dissolved aminobenzoic acid in the fermentation reactor, the possibility of crystallizing the majority of the aminobenzoic acid with carbon dioxide, and a saving of base in the fermentation.

[0010] When aminobenzoic acid is separated by filtration, a mother liquor remains that still contains a significant residual concentration of aminobenzoic acid (corresponding to the solubility of aminobenzoic acid under the respective conditions). Therefore, this dissolved aminobenzoic acid should be removed as quantitatively as possible before being disposed of as wastewater. In addition, the mother liquor usually contains dissolved residual components of usable media components, such as carbon or nitrogen sources, which were not fully converted to aminobenzoic acid or biomass during fermentation. The effort required to remove these usable media components from the mother liquor usually exceeds the economic benefit, which is why disposal with wastewater is preferred in the current state of the art.

[0011] For the recovery of aminobenzoic acid from dilute aqueous solutions, various concepts have been developed in the prior art, which are described in WO 2023 / 117756 A1 on pages 3 to 5. As explained therein, none of these concepts is without disadvantages. WO 2023 / 117756 A1 itself proposes extracting the aqueous mother liquor containing dissolved aminobenzoic acid obtained in the process described therein with an alkanol having 8 to 12 carbon atoms. Then, the aminobenzoic acid dissolved in the alkanol phase is recovered by basic or acidic back-extraction in the form of an aqueous solution of an aminobenzoic acid salt, and finally, precipitating it from the salt solution by adjusting the pH to 3.0 to 4.7.Although the use of Cs-C alkanols as extraction agents in conjunction with basic or acidic back-extraction represents a good compromise between the requirement for the most complete extraction of the aminobenzoic acid from the mother liquor (to achieve the highest possible yield) on the one hand and the requirement to obtain the purest possible extracted mother liquor (to simplify wastewater treatment and disposal) on the other, this process is not without its drawbacks. The necessity of extracting the entire mother liquor involves considerable effort. Even more serious, however, is that this application also does not offer a fundamental solution to the problem of high salt loads in wastewater.In the previously mentioned application WO 2019 / 234092 A1, it is proposed to recycle the mother liquor remaining after separation of the aminobenzoic acid precipitated in the carbon dioxide crystallization to the ion exchange step. Furthermore, it is proposed to crystallize further aminobenzoic acid from the aqueous fermentation solution remaining after separation of the calcium aminobenzoate produced in the fermentation by adding a mineral acid. The additional mother liquor remaining after separation of the aminobenzoic acid precipitated in this process can be subjected to an adsorption step to recover any residual aminobenzoic acid dissolved therein, and adsorbed aminobenzoic acid can be recovered by a subsequent desorption step.Depending on the pH at which desorption is carried out, the resulting desorbate can be recycled to the ion exchange step (desorption at pH values ​​from 6.0 to 11.0) or subjected to post-crystallization by base addition (desorption at pH values ​​below 3.0). The recycling of mother liquor (regardless of its origin) to the fermentation is not disclosed in WO 2019 / 234092 A1.

[0012] There was therefore a need for further improvements in the field of fermentative production of aminobenzoic acid. In particular, it would be desirable to reduce the salt contamination of the wastewater and the yield losses via the mother liquor.

[0013] In response to this need, the present invention provides a process comprising the preparation of aminobenzoic acid, the process comprising the following steps:

[0014] (A) Providing a fermentation broth containing aminobenzoate anions, comprising: (A1) fermenting a fermentable carbon-containing compound and a nitrogen-containing compound in the presence of microorganisms selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans or a mixture of two or more of the aforementioned microorganisms at a pH of > 5.5 and (A.2) separating the microorganisms;

[0015] (B) adjusting the pH of the fermentation broth containing aminobenzoate anions to 3.0 to 5.5, preferably 3.5 to 4.5, particularly preferably 3.8 to 4.2, most preferably 4.0, by adding formic acid, whereby aminobenzoic acid precipitates;

[0016] (C) separating aminobenzoic acid precipitated in step (B) to obtain a mother liquor containing formate anions and formic acid; and

[0017] (D) introducing 55.0% to 100% of the total mother liquor obtained in step (C) (containing formate anions and formic acid), optionally after treatment (such as in particular sterile filtration and / or thermal inactivation), into the fermentation according to step (A1) with depletion of the formate anions and formic acid by

[0018] (i) an added enzyme preparation, and / or

[0019] (ii) the microorganisms used in step (A) and / or

[0020] (iii) added further microorganisms which are different from the microorganisms used in step (A)

[0021] (by oxidizing the formate anions and the formic acid to carbon dioxide and / or using them as a substrate for biomass production by the microorganisms from step (A) and / or the aminobenzoate anions and / or using them as a substrate for biomass production by the further microorganisms).

[0022] Completely surprisingly, it was discovered that the use of formic acid in the crystallization of aminobenzoic acid leads to the formation of a mother liquor that can be returned to the fermentation without an accumulation of formate anions or formic acid occurring there. When using formic acid (and not strong mineral acids such as hydrochloric acid, as is customary in the prior art) as the agent for the crystallization of aminobenzoic acid, a mother liquor is obtained which contains the formate anions formed by protonation of the aminobenzoate anions. In addition, the mother liquor also contains free formic acid, since formic acid, unlike HCl, does not completely decompose under the existing conditions with the formation of H3O. +ions are dissociated. For the sake of simplicity, the following description refers to a "mother liquor containing formate anions"; this formulation includes the presence of free formic acid. Formic acid, whether present as formate anion or free acid, is oxidized to carbon dioxide during fermentation, incorporated into the target product of the fermentation, the aminobenzoate anions, and / or metabolized to form biomass. This is achieved by the microorganisms used in step (A) or, if these are not capable of doing so or not capable of doing so to a sufficient extent, by added enzyme preparations or by added further microorganisms different from those used in step (A) that are capable of doing so. These possibilities can also be combined. Further below, it is explained in more detail how suitable microorganisms and enzyme preparations can be provided.

[0023] It was also discovered, quite surprisingly, that returning the mother liquor containing formate anions to the fermentation, despite its comparatively low pH in the range of 3.0 to 5.5 (see step (B) above), does not require the addition of more base to maintain the desired pH for the fermentation. Quite the opposite, it was found that even less base, ideally no base at all, is required to adjust the pH. Without wishing to be bound by any theory, it is assumed that formate degradation during fermentation occurs at least partially with the release of carbon dioxide from the fermentation broth, and that this release of "carbonic acid" leads to an increase in pH, which compensates for the initial pH reduction caused by the introduction of the acidic mother liquor and, ideally, even further compensates for the pH reduction caused by the formation of aminobenzoic acid.

[0024] By recycling the mother liquor containing formate anions, other dissolved components, such as unreacted carbon or nitrogen sources, as well as unreacted nutrients such as salts, trace elements, and vitamins, are also returned to the fermentation. Furthermore, the portion of aminobenzoic acid still dissolved in the mother liquor is recycled, eliminating the additional effort required to extract the dissolved aminobenzoic acid from the mother liquor for the recycled portion.

[0025] In the context of the present invention, all pH values ​​refer to the temperature at which the corresponding step (e.g. step (A1) or (B)) is carried out and can be easily measured with a glass electrode.

[0026] First, a brief summary of various possible embodiments of the invention follows:

[0027] In a first embodiment of the invention, which can be combined with all other embodiments, water is (partially) separated, in particular evaporated, after step (A.2) and before step (B) or after step (B) and before step (C). In a second embodiment of the invention, which can be combined with all other embodiments, the nitrogen-containing compound is selected from ammonia gas, ammonia water, (at least) one ammonium salt, soy protein, urea, or a mixture of two or more of the aforementioned nitrogen-containing compounds.

[0028] In a third embodiment of the invention, which can be combined with all other embodiments, the fermentable carbon-containing compound is selected from starch hydrolysate, an alkali metal or ammonium formate, sugar cane juice, sugar beet juice, hydrolysates from lignocellulose-containing raw materials or a mixture of two or more of the aforementioned carbon-containing compounds.

[0029] In a fourth embodiment of the invention, which can be combined with all other embodiments, provided that they do not exclude the use of further microorganisms different from those used in step (A), the further microorganisms are selected from Pichia pastoris, Hansenula polymorpha, or a mixture of both.

[0030] In a 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, particularly preferably 6.6 to 8.0, is maintained in step (A1).

[0031] In a sixth embodiment of the invention, which can be combined with all other embodiments, the 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, based on the total mass of the mixture, is at least 20%, preferably 60% to 98%, particularly preferably 70% to 95%.

[0032] In a seventh embodiment of the invention, which can be combined with all other embodiments except those limited to the formation of meta- or para-aminobenzoic acid, ortho-aminobenzoic acid is prepared.

[0033] In an eighth embodiment of the invention, which can be combined with all other embodiments except those limited to the formation of ortho- or meta-aminobenzoic acid, para-aminobenzoic acid is prepared.

[0034] In a ninth embodiment of the invention, which is a particular embodiment of the seventh embodiment, the ortho-aminobenzoic acid obtained in step (C) is converted into a poly(anthranilamide).

[0035] In a tenth embodiment of the invention, which is a further particular embodiment of the seventh embodiment, the ortho-aminobenzoic acid obtained in step (C) is converted into an anthranilic acid derivative selected from anthranilic acid halide, isatoic anhydride, or a mixture thereof, and the anthranilic acid derivative is reacted with a polyol to form a polyamine. In an eleventh embodiment of the invention, which is a particular embodiment of the tenth embodiment, the polyamine is phosgenated to form a polyisocyanate.

[0036] In a twelfth embodiment of the invention, which can be combined with all other embodiments except those which exclude the formation of aniline from the aminobenzoic acid, and which can be advantageously combined in particular with the seventh embodiment, the aminobenzoic acid from step (C) is converted to aniline with elimination of carbon dioxide.

[0037] In a thirteenth embodiment of the invention, which is a particular embodiment of the twelfth embodiment, the aniline is reacted with formaldehyde to form methylenediphenylenediamine and polymethylenepolyphenylenepolyamine.

[0038] In a fourteenth embodiment of the invention, which is a particular embodiment of the thirteenth embodiment, the methylenediphenylenediamine and / or the polymethylenepolyphenylenepolyamine is phosgenated to methylenediphenylene diisocyanate and / or polymethylenepolyphenylene polyisocyanate.

[0039] In a fifteenth embodiment of the invention, which is a particular embodiment of the fourteenth embodiment, the methylenediphenylene diisocyanate and / or the polymethylenepolyphenylene polyisocyanate is reacted with a polyol to form a polyurethane.

[0040] In a sixteenth embodiment of the invention, which is a further particular embodiment of the twelfth embodiment, the aniline is converted to an azo compound.

[0041] In a seventeenth embodiment of the invention, which is a particular embodiment of the twelfth to sixteenth embodiments, the carbon dioxide formed in the reaction to aniline is converted to formic acid, and the formic acid thus obtained is used in step (B).

[0042] In an eighteenth embodiment of the invention, which can be combined with all other embodiments, carbon dioxide optionally formed in the fermentation in step (A1) (in particular by oxidation of the formate anions and / or the formic acid) is converted to formic acid, and the formic acid thus obtained is used in step (B).

[0043] In a nineteenth embodiment of the invention, which is a particular embodiment of the seventeenth and eighteenth embodiments, the conversion to formic acid is carried out by hydrogenation or acid electrolysis of the carbon dioxide.

[0044] In a twentieth embodiment of the invention, which can be combined with all other embodiments, in step (D) the first part of the mother liquor is admixed with a further carbon-containing compound, a further nitrogen-containing compound, (at least) one compound for reducing foam formation in the 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 iron sulfate, manganese sulfate, copper sulfate, zinc sulfate or nickel chloride, or a mixture of two or more of the aforementioned compounds, before being introduced into the fermentation.

[0045] In a twenty-first embodiment of the invention, which can be combined with all other embodiments, in step (D) less than 100%, in particular up to 99.5%, of the total mother liquor obtained in step (C) is introduced into the fermentation according to step (A1), wherein the part of the mother liquor not fed into the fermentation is fed to a wastewater treatment plant after depletion of aminobenzoic acid.

[0046] In a twenty-second embodiment of the invention, which is a particular embodiment 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.

[0047] In a twenty-third embodiment of the invention, which is a particular embodiment of the twenty-second embodiment, the organic solvent comprises an alkanol having 8 to 12, preferably 9 to 11 carbon atoms, in particular 1-dodecanol, and in which the adsorbent comprises activated carbon or a polymeric adsorbent, in particular based on polystyrene and / or polydivinylbenzene.

[0048] In a twenty-fourth embodiment of the invention, which can be combined with all other embodiments, provided that they do not exclude the use of enzyme preparations, the enzyme preparation contains a formate dehydrogenase and / or a formate oxidase.

[0049] In a twenty-fifth embodiment of the invention, which is a particular embodiment of the twenty-fourth embodiment, the formate dehydrogenase belongs to EC class 1.17.1.9 and the formate oxidase belongs to EC class 1.2.3.1.

[0050] The embodiments briefly described above and further possible embodiments of the invention are explained in more detail below. All of the embodiments described above and the further embodiments of the invention described below can be combined with one another as desired, unless the context clearly indicates the opposite to a person skilled in the art or unless expressly stated otherwise. PREPARATION OF THE FERMENTATION BROTH CONTAINING AMINO BENZOATE ANIONS (STEP (A))

[0051] Step (A) of the process comprises at least steps (A1), the fermentation of a fermentable carbon-containing compound and a nitrogen-containing compound in the presence of microorganisms, and (A.2) the separation of the microorganisms. The fermentation is carried out in a dedicated reaction apparatus, the fermentation reactor. The reaction mixture present in the fermentation reactor is referred to as the fermentation broth. The fermentation in step (A1) is carried out such that the pH in the fermentation broth is greater than 5.5 and preferably in the range from 5.6 to 11, more preferably in the range from 6.0 to 8.0, and most preferably in the range from 6.6 to 8.0. If necessary, the pH can be regulated by adding a base, in particular by adding aqueous or gaseous ammonia, aqueous potassium hydroxide, or aqueous sodium hydroxide.At these pH values, the aminobenzoic acid is present predominantly or entirely in the form of its anion (the aminobenzoate anion, H2NC6H4COO-); therefore, in the context of the present invention, the fermentation broth is referred to as containing aminobenzoate anions. (The nature of the counterion depends on the exact conditions; in particular, Na + , K + and / or NH4 + (It was found, quite surprisingly, that this addition of a base for pH control in the process according to the invention can be reduced as a result of the recycling of the mother liquor containing formate anions and, ideally (apart from special situations such as, for example, the start-up of the process) can be omitted altogether.

[0052] Preferred microorganisms for carrying out step (A1) are prokaryotes (such as, in particular, bacteria). Suitable microorganisms include, for example, Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, and Bacillus coagulans. Escherichia coli, Pseudomonas putida, and / or Corynebacterium glutamicum are preferably used. The use of mixtures of different microorganisms is possible, but the use of microorganisms of a single species is preferred. The sole use of Corynebacterium glutamicum, in particular Corynebacterium glutamicum ATCC 13032, is particularly preferred.In particular, reference is made to patent applications WO 2015 / 124686 A1 and WO 2015 / 124687 A1, which describe fermentation processes using bacteria (see, for example, WO 2015 / 124687 A1, page 15, line 8 to page 16, line 30, Example 1 (page 29, lines 4 to 26), Example 3 (especially page 34, lines 10 to 18) and Example 4 (especially page 55, lines 9 to 31). In particular, bacteria are used that are able to convert a fermentable carbon-containing compound into aminobenzoate anions in the presence of a suitable nitrogen source without these being immediately consumed in intracellular biochemical processes, so that aminobenzoate anions accumulate in the cell and ultimately pass into the fermentation broth. To obtain such prokaryotes, two fundamental Ways are available, which can also be combined in a preferred design:.

[0053] (i) The enzymatic reactions in the aminobenzoic acid pathway of the prokaryotic cell can be increased so that aminobenzoic acid is produced faster than it is consumed.

[0054] (ii) The subsequent reactions by which aminobenzoic acid is converted into further metabolites or products (e.g. tryptophan) can be reduced or eliminated, resulting in an accumulation of aminobenzoic acid in the cell.

[0055] Methods for obtaining prokaryotic organisms with the aforementioned properties are known in the art. Suitable prokaryotes can be identified, for example, by screening for mutants that release aminobenzoic acid into the surrounding medium. However, the targeted modification of key enzymes using genetic engineering techniques is preferred. Gene expression and enzyme activity can be enhanced, reduced, or even completely suppressed using conventional genetic engineering methods. This results in recombinant strains. A preferred embodiment for the particularly preferred ortho-isomer is described below (for the para-isomer, see, for example, T. Kubota et al., Metabolie Engineering 2016, 38, 322-330 ("Production of para-aminobenzoate by genetically engineered Corynebacterium glutamicum and non-biological formation of an N-glucosyl byproduct" [1]):

[0056] Particularly preferably, the prokaryotes capable of converting a fermentable carbon-containing compound into aminobenzoic acid in the presence of a nitrogen-containing compound contain a modification of the anthranilate phosphoribosyltransferase activity, which reduces said enzyme activity. This modification reduces or completely prevents the conversion of ortho-aminobenzoate to N-(5-phospho-D-ribosyl)-anthranilate. This results in an accumulation of aminobenzoic acid in the cell. The term "anthranilate phosphoribosyltransferase activity" refers to an enzyme activity that catalyzes the conversion of ortho-aminobenzoate to N-(5-phospho-D-ribosyl)-anthranilate.

[0057] In the bacterium Corynebacterium glutamicum, the anthranilate phosphoribosyltransferase activity is encoded by the trpD gene (cg3361, Cgl3032, NCgl2929). In Pseudomonas putida, encoding occurs via the trpD gene (PP_0421) within the trpDC operon.

[0058] The described reduction of anthranilate phosphoribosyltransferase activity can in principle be achieved in three ways:

[0059] (i) The regulation of expression of the gene for anthranilate phosphoribosyltransferase activity can be modified so as to reduce or abolish transcription of the gene or subsequent translation.

[0060] (ii) The nucleic acid sequence of the gene for anthranilate phosphoribosyltransferase activity can be modified so that the enzyme encoded by the modified gene has a lower specific activity.

[0061] (iii) The native gene for anthranilate phosphoribosyltransferase activity can be replaced by another gene derived from a different organism and encode an enzyme with a specific anthranilate phosphoribosyltransferase activity lower than that of the previously mentioned native genes (e.g., TRP4, trpD or trpDC).

[0062] In a preferred embodiment of the present invention, cells of Corynebacterium glutamicum, preferably C. glutamicum ATC13032, are used as microorganisms for preparing a fermentation broth containing aminobenzoate anions. These preferably contain the modifications defined below. Further details on this strain and its metabolic activities are disclosed in WO 2023 / 111055.

[0063] (i) An anthranilate phosphoribosyltransferase activity that is reduced compared to the respective wild type. In one embodiment, no corresponding enzyme activity is present. Genetic modifications with which this can be achieved are generally known. These include the deletion of the gene in question or a modification of the coding region of the gene so that a truncated or otherwise inactive enzyme is expressed. In an alternative embodiment, the activity of the enzyme is reduced compared to the activity present in the wild type, although residual activity must still be present. This residual activity is preferably between 10% and 60%, more preferably between 20% and 50% of the activity present natively in C. glutamicum ATCC13032.This is preferably achieved by reduced expression of the gene for anthranilate phosphoribosyltransferase (trpD) compared to the wild type, although expression is not completely suppressed. This is preferably done by using a promoter sequence that has lower transcriptional activity than the endogenously present promoter sequence or by modifying the distance between the ribosome binding site and the start codon of the trpD gene or by changing the start codon itself. In a preferred embodiment of the present invention, the activity of anthranilate phosphoribosyltransferase is reduced by deleting or inactivating the gene for endogenous anthranilate phosphoribosyltransferase (trpD) and replacing this gene with a gene for an anthranilate phosphoribosyltransferase with a modified ribosomal binding site and optionally a modified start codon as in SEQ ID NO. 2 or 6, preferably SEQ ID NO.6. The amino acid sequence of the anthranilate phosphoribosyltransferase preferably corresponds to the endogenous anthranilate phosphoribosyltransferase, particularly preferably it is defined by SEQ ID NO. 7 or a variant thereof.

[0064] (ii) Increased activity of shikimate kinase. This is preferably achieved by increased expression of a corresponding enzyme. In one embodiment of the present invention, the increase in activity is achieved by increased expression of the gene for the endogenously present shikimate kinase as defined in SEQ ID NO. 8 or a variant thereof. In another preferred embodiment, this is achieved by expression of an exogenous shikimate kinase, preferably as defined in SEQ ID NO. 9 or a variant thereof. Increased expression of a gene can be achieved by any method known to the person skilled in the art, in particular by introducing multiple copies of the corresponding gene into the microorganism or by using stronger promoters to express the endogenously present enzyme. A particularly preferred promoter for the expression of foreign genes or the increased expression of endogenous genes is Ptu / as defined in SEQ ID NO. 10.

[0065] (iii) Increased activity of 3-phosphoshikimate-l-carboxyvinyltransferase and chorismate synthase. These enzymes preferably have an amino acid sequence as 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 additional copies of the genes encoding these enzymes into the microorganism. The Ptu / promoter is preferably used to control expression.

[0066] (iv) Advantageous, but not obligatory, is the presence of a 3-deoxyarabinoheptulosate 7-phosphate synthase (DAHP synthase) which is feedback-resistant, i.e. not inhibited by its product or by a product resulting from the product. Preference is given to an enzyme having the amino acid sequence defined in SEQ ID NO. 13 or a variant thereof.

[0067] The person skilled in the art is aware that, based on the known metabolic pathway leading to ortho-aminobenzoic acid in C. glutamicum, further modifications can be introduced to increase the efficiency of the strain described above.

[0068] In another preferred embodiment of the present invention, cells of Escherichia coli, preferably E. coli K12, are used as microorganisms for providing a fermentation broth containing aminobenzoate anions, as disclosed in WO 2022 / 090363.

[0069] Particularly preferred is a strain of E. coli which expresses an anthranilate phosphoribosyltransferase (TrpD) and a glutamine amidotransferase (TrpG).

[0070] Preferred is the TrpG domain as defined by amino acid positions 3 to 196 of SEQ ID NO. 14 (TrpGD from E. coli) or a variant thereof. Also preferred are TrpG from Bacillus subtilis (SEQ NO. 15), the TrpG domain of TrpGD from Salmonella typhimurium (amino acid positions 3 to 196 of SEQ ID NO. 16), TrpG from Cupriavidus necator (SEQ ID NO. 17), TrpG from Corynebacterium glutamicum (SEQ ID NO. 18), or a variant of one of the aforementioned polypeptides.

[0071] Preferred is the TrpD domain as defined by amino acid positions 202 to 531 of SEQ ID NO. 14 (TrpGD from E. coli) or a variant thereof. Also preferred are TrpD from Bacillus subtilis (SEQ NO. 19), the TrpD domain of TrpGD from Salmonella typhimurium (amino acid positions 202 to 531 of SEQ ID NO. 16), TrpD from Cupriavidus necator (SEQ ID NO. 20), TrpD from Corynebacterium glutamicum (SEQ ID NO. 21), or a variant of one of the aforementioned polypeptides.

[0072] When TrpG and TrpD are expressed separately, it is preferred that the expression of TrpD is lower than the expression of TrpG. Preferably, it is at least 10%, more preferably at least 20%, even more preferably at least 40%, and most preferably at least 60% lower. However, it is particularly preferred that a minimal expression of TrpD is maintained, which is at least 5% of the expression of TrpG. The expression of the genes encoding the aforementioned proteins is preferably determined at the mRNA level by quantitative TR-PCR. Such different expressions of the two polypeptides are preferably achieved by introducing the genes encoding them into the cell in different expression cassettes under the control of promoters of different strengths. Alternatively, the introduction of the respective genes in different copy numbers is also possible.The same promoter can then be used for the expression of both polypeptides.

[0073] In yet another embodiment, cells of Pseudomonas putida, preferably P. putida KT2440, are used as microorganisms to provide a fermentation broth containing aminobenzoate anions. Genetic modifications that enable this bacterium to synthesize ortho-aminobenzoic acid are described in Example 4 of WO 2015 / 124687.

[0074] The depletion of formate anions and formic acid in step (A) is preferably carried out by microorganisms or an enzyme preparation. It is also possible to combine microorganisms with an enzyme preparation.

[0075] In one embodiment of the present invention, the microorganisms used to deplete formate anions and formic acid are the same microorganisms used to prepare the fermentation broth containing aminobenzoate anions. In many cases, these microorganisms already express the required enzymes natively, i.e., without further genetic modification. For example, formate dehydrogenases are known for some strains of Escherichia coli (Sawers, 1994, "The hydrogenases and formate dehydrogenases of Escherichia coli", Antonie van Leeuwenhoek, 66: 57-88). For Corynebacterium glutamicum (Witthof et al., 2012, "Corynebacterium glutamicum harbors a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate", Microbiology, 158: 2428-2439) and Pseudomonas putida (Roca et al., 2009, "Redundancy of Enzymes for Formaldehyde Detoxification in Pseudomonas putida", Journal of Bacteriology, 191: 3367-3374). If the microorganisms used lack the enzymes to degrade formate anions or formic acid, or if the native enzyme activity is insufficient, the corresponding capabilities of the microorganisms can be increased using generally known genetic engineering methods. This can be done by (i) introducing foreign genes, in particular those encoding the formate dehydrogenases or oxidases described below, into the microorganisms, (ii) introducing additional copies of the native genes for the corresponding enzymes, or (iii) replacing the native promoters of the natively present genes for the corresponding enzymes with stronger promoters.

[0076] In a further embodiment, additional microorganisms are used that are not suitable for producing aminobenzoic acid. In this case, the fermentation broth contains at least two types of microorganisms: (i) those used to produce the aminobenzoic acid through fermentation and (ii) those that serve solely to deplete formate anions and formic acid ("additional microorganisms"). In one embodiment, the additional microorganisms used for depleting formate anions and formic acid are those that already natively express the corresponding enzymes in sufficient quantities. In another embodiment, the additional microorganisms used are those whose corresponding enzyme activities were generated or enhanced using the methods for the embodiment defined in the preceding paragraph.If additional microorganisms are used, so that the fermentative production of aminobenzoic acid and the depletion of formate anions and formic acid are separated, it is not necessary for the additional microorganisms to still be capable of replication. Therefore, in one embodiment of the present invention, additional microorganisms that are no longer capable of replication can be used. This can be achieved, for example, by using a strain with auxotrophy that is not compensated for in the fermentation for aminobenzoic acid production. Alternatively or additionally, heating is also possible, provided the additional microorganisms are sufficiently temperature-stable.

[0077] The term "enzyme preparation" refers to compositions containing (at least) one formate dehydrogenase and / or (at least) one formate oxidase and / or (at least) one formate hydrogen lyase, with the presence of formate dehydrogenases and / or formate oxidases being preferred. Preferred formate dehydrogenases and oxidases are described below in this application. According to the invention, it is not necessary for the formate dehydrogenase or oxidase to be present in pure form. Therefore, in one embodiment of the present invention, the enzyme preparation is a crude extract of cells, preferably microorganisms, that express the enzyme in question. In a further embodiment, the enzyme preparation contains or consists of at least one purified formate oxidase or dehydrogenase.

[0078] For the depletion of formate anions or formic acid, formate dehydrogenases, preferably of EC class 1.17.1.9, or formate oxidases, preferably of EC class 1.2.3.1, are preferably used.

[0079] Formate dehydrogenases as defined by SEQ ID NO. 22 or 23, as well as variants of these enzymes, are preferred. Formate oxidases are the enzymes defined by SEQ ID NO. 24 or 25, as well as their variants.

[0080] In the present application, "variant" is understood to mean a polypeptide obtained by adding, deleting or exchanging up to 20%, preferably up to 15%, more preferably up to 10% and most preferably up to 5% of the amino acids contained in the respective polypeptide. In principle, the aforementioned modifications can be made continuously or discontinuously at any desired position in the polypeptide. However, they preferably only occur at the N-terminus and / or the C-terminus of the polypeptide. Substitutions of amino acids are preferably conservative substitutions, i.e. those in which the modified amino acid has a residue with similar chemical properties to the amino acid present in the unaltered polypeptide.Thus, amino acids with basic residues are particularly preferably exchanged for those with basic residues, amino acids with acidic residues for those with acidic residues, amino acids with polar residues for those with polar residues, and amino acids with nonpolar residues for those with nonpolar residues. The specific enzyme activity of a variant of one of the polypeptides defined above is preferably at least 80% of the specific activity of the unaltered polypeptide. Enzyme tests for detecting the activity of the aforementioned enzymes can be found in the literature by those skilled in the art.

[0081] Aminobenzoic acid occurs in three isomeric forms (ortho-, meta-, and para-aminobenzoic acid). In principle, the process can be applied to all three isomers, either in isomerically pure form or as mixtures of different isomers. However, preference is given to the preparation of ortho-aminobenzoic acid or para-aminobenzoic acid, especially in isomerically pure form. Particular preference is given to the preparation of ortho-aminobenzoic acid, especially in isomerically pure form. "Isomerally pure" in this context means that the molar fraction of the desired aminobenzoic acid isomer, based on all aminobenzoic acid isomers present, is at least 99.0 mol%, preferably at least 99.9 mol%, particularly preferably 100 mol%. As is known in the art, the formation of the desired isomer can be controlled enzymatically. For example, in the shikimate pathway, chorismate can be enzymatically converted to anthranilate (= anion of ortho-aminobenzoic acid).Alternatively, there are also enzyme-catalyzed reactions from chorismate to para-aminobenzoate (= anion of para-aminobenzoic acid).

[0082] Regardless of which microorganism is used and which isomer is desired, the fermentation broth at the beginning of the fermentation in step (A1) comprises recombinant cells of the microorganism used and at least one fermentable carbon-containing compound (as well as at least one nitrogen-containing compound as a nitrogen source). Preferably, the fermentation broth also contains further components selected from the group consisting of buffer systems, inorganic nutrients, amino acids, vitamins, and other organic compounds required for the growth or maintenance metabolism of the recombinant cells. The fermentation broth is water-based. After the start of the fermentation process, the fermentation broth also comprises aminobenzoate anions, the desired fermentation product.

[0083] As already mentioned, a fermentable carbon-containing compound within the meaning of the present invention is understood to be any organic compound or mixture of organic compounds that can be used by the recombinant cells of the microorganism employed to produce aminobenzoate anions. The production of the aminobenzoate anions can take place in the presence or absence of oxygen. Production in the presence of oxygen is preferred, in particular by introducing an oxygen-containing gas such as air.

[0084] Preferred are fermentable carbon-containing compounds that can also serve as an energy and carbon source for the growth of the recombinant cells of the microorganism used. Suitable compounds are starch hydrolysate, alkali metal or ammonium formates, sugar cane juice, sugar beet juice, and / or hydrolysates from lignocellulose-containing raw materials, with starch hydrolysate, sugar cane juice, sugar beet juice, and / or hydrolysates from lignocellulose-containing raw materials being preferred. Ammonia gas, ammonia water, (at least) one ammonium salt, soy protein, and / or urea are preferably used as the nitrogen source.

[0085] In one embodiment, step (A1) is carried out continuously, i.e. the reactants are continuously fed to 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 the aqueous fermentation broth containing aminobenzoate anions, including the microorganisms present therein. The microorganisms can be separated outside the fermentation reactor by known separation processes, such as, in particular, filtration, centrifugation or sedimentation. However, it is also conceivable to retain the microorganisms in the fermentation reactor by using known separation processes (in particular filtration) and to remove a clarified fermentation broth containing aminobenzoate anions from the latter. In this embodiment, therefore, step (A.2), the separation of the microorganisms is already carried out in the fermentation reactor itself.

[0086] In another embodiment, step (A1) is carried out in a discontinuous process (batch mode) in fermentation cycles. A fermentation cycle preferably comprises the initial introduction or addition of microorganisms into a nutrient medium, the introduction and / or addition of nutrients, the build-up of microorganisms, the formation of the desired product, i.e. the aminobenzoate anions, and the complete or partial emptying of the reactor after completion of the fermentation. In a variant of the discontinuous process (so-called "fed-batch process"), the reactants are fed to the fermentation reactor (continuously or discontinuously [i.e.in portions]), optionally after prior sterilization by filtration or thermal treatment, as the reactor volume allows, without products - with the exception of gaseous components which are discharged via a connection of the fermentation reactor to an exhaust system - being removed from the fermentation reactor. The reaction is interrupted after the maximum possible amount of reactants has been added and the product mixture is removed from the fermentation reactor. In the case of a batch process, clarification of the fermentation broth, in particular by filtration, centrifugation or sedimentation, outside the fermentation reactor is preferred. Before the fermentation broth is returned, sterilization by filtration or thermal treatment can optionally be carried out, if necessary.

[0087] Separated microorganisms (biomass) can be returned to the fermentation, possibly minus a discharged portion.

[0088] Regardless of the precise operating mode, the fermentation reactor preferably includes devices for measuring important process parameters such as temperature, pH, substrate and product concentration, 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 aforementioned process parameters.

[0089] Suitable fermentation reactors include stirred tank reactors, membrane reactors, or loop reactors. Stirred tank reactors and loop reactors are particularly preferred for both aerobic and anaerobic fermentations (preferably airlift reactors, in which the circulation of the liquid in the reactor is achieved by gassing).

[0090] In addition to clarification (step (A.2)), the fermentation broth from step (A1) can be subjected to further pretreatment steps before being fed to step (B). Of particular note here is decolorization of the fermentation broth (optional step (A.3)). Such decolorization is preferably carried out by passing the fermentation broth, freed from microorganisms, over a column with a fixed packing to remove colorants by adsorption. Diatomaceous earth or an ion exchange packing, for example, can be used as a possible solid phase. Such decolorization is preferably carried out when the fermentation broth contains colored substances that could interfere with the subsequent crystallization in step (B).

[0091] Crystallization and isolation of aminobenzoic acid from the fermentation broth containing aminobenzoate anions (step (B) and step (C))

[0092] In step (B) of the process, the aminobenzoic acid is precipitated (crystallized) from the fermentation broth containing aminobenzoate anions. This step is carried out in a technical apparatus suitable for crystallization, known in the art as a crystallizer.

[0093] Suitable crystallizers are, for example, stirred tanks or forced circulation crystallizers such as those of the "Oslo type". In the crystallizer, the pH is adjusted to values ​​in the range from 3.0 to 5.5, preferably 3.5 to 4.5, particularly preferably 3.8 to 4.2, very particularly preferably 4.0. This is done by adding formic acid, either in pure form (anhydrous) or preferably in a mixture with water. If a mixture of formic acid and water is used, the mass concentration of formic acid in this mixture, based on the total mass of the mixture, is preferably at least 20%, particularly preferably 60% to 98%, and very particularly preferably 70% to 95%. Through this pH adjustment, the aminobenzoic acid anions (H2NC6H4COO") are predominantly or completely converted into the electroneutral form (H2NC6H4COOH or HsN CgH^OO") and crystallize out. This type of crystallization is also called reactive crystallization.The crystallized aminobenzoic acid is then separated in step (C); this can be done by known methods such as, in particular, filtration, sedimentation or centrifugation, leaving behind the mother liquor containing formate anions and containing dissolved portions of aminobenzoic acid.

[0094] Depending on the concentrations present, it may be useful to separate off some of the water present to facilitate the isolation of the aminobenzoic acid in step (C) and / or to prevent the volume of mother liquor fed into the fermentation from becoming too large. Such a step can be carried out before (i.e. after step (A.2) and before step (B)) or after (i.e. after step (B) and before step (C)), preferably before, the crystallization in step (B). This separation of water can be achieved by evaporation or by means of a membrane process. All evaporation apparatus known in the art are suitable for evaporation. To minimize thermal stress, it is preferable to carry out the evaporation at reduced pressure, in particular at 0.1 mbar to 900 mbar, particularly preferably at 100 mbar to 500 mbar.This enables gentle evaporation of water at temperatures of 45 °C to 97 °C, in particular up to 82 °C, at which decarboxylation of aminobenzoic acid or, if the evaporation takes place after step (B), the decomposition of formic acid can be almost completely prevented.

[0095] It has proven useful to feed the fermentation broth and the formic acid to the crystallizer via feed devices that are spatially (as far as possible) separated from one another. This ensures that the reactants are mixed as thoroughly as possible with the reactor contents before the acid-base reaction occurs. Suitable feed devices include, for example, pipelines, preferably with shut-off valves. In one embodiment, the feed device for the fermentation broth and the feed device for the acid are arranged at opposite points on the reactor wall (essentially) at right angles to the latter. In another embodiment, the feed device for the fermentation broth and the feed device for the acid are arranged (essentially) parallel to the reactor wall, with the feed devices being opposite one another and located as close as possible, in particular directly, to the reactor wall.

[0096] It is possible to divide the crystallizer into chambers using suitable internal fittings. The flow direction can be adjusted by selecting the stirrer geometry and operating mode. It is also possible to equip the crystallizer with an external pumping circuit, in which case one of the two reactants—fermentation broth or formic acid—is fed into the pumping circuit and the other directly into the crystallizer. If a crystallizer is operated with a classifier and a pumping circuit, the pumping circuit is installed at the bottom of the classifier for fluidization or at the side of the classifier.

[0097] Crystallization in the crystallizer can be carried out continuously or batchwise. Continuous operation is preferred. Regardless of the operating mode (continuous or batchwise), the exact operating parameters are determined (among other things) by the desired crystal size, which can be adjusted by the residence time / reaction time and the degree of supersaturation (large crystal sizes are favored by long residence times / long reaction times and low degrees of supersaturation).

[0098] Crystallization is preferably carried out in the presence of seed crystals:

[0099] In a batch crystallization, the preferred procedure is to first place the fermentation broth in the crystallizer and heat it to a defined temperature (preferably 5 °C to 40 °C, for example 20 °C). If the pH of the fermentation broth is significantly above the solubility limit of aminobenzoic acid at the selected temperature, the fermentation broth is first slightly acidified with formic acid to a pH that corresponds to or is at least close to the solubility limit of aminobenzoic acid at the selected temperature and under the given boundary conditions (preferably pH 5.0 to 6.5). This slight acidification can be carried out quickly. Seed crystals of the desired polymorph of aminobenzoic acid are then added (in the case of anthranilic acid, this is preferably polymorph (form) I).This polymorph has comparatively low solubility and therefore promotes the most complete recovery of the aminobenzoic acid possible. The amount of seed crystals added is preferably approximately 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). The pH is then adjusted to 3.0 to 5.5, preferably 3.5 to 4.5, particularly preferably 3.8 to 4.2, and most preferably 4.0, by adding further formic acid. The formic acid is preferably added slowly; for example, with 1 kg of fermentation broth initially charged and 75% formic acid used, the addition takes 1 h. After the acid addition is complete, stirring is continued for a certain period, in particular for the same period of time that the addition of the acid after the addition of the seed crystals took.The precipitated aminobenzoic acid is then separated, in particular by filtration (if necessary under vacuum), sedimentation, or centrifugation (preferably by centrifugation), and preferably washed several times (in particular twice) with an aqueous, in particular acidic, preferably formic acid, washing liquid having a pH of 3.0 to 5.5, preferably 3.0 to 4.5, particularly preferably 3.2 to 3.8, most preferably 3.5. Depending on the purity requirements of the intended subsequent application, the aminobenzoic acid can also be purified by recrystallization.

[0100] In continuous crystallization, seed crystals generally only need to be added specifically during the start-up of the continuous process, since additional seed crystals later form spontaneously in situ (so-called secondary nucleation) or are available in sufficient quantities. The suspension of seed crystals required for start-up can be prepared as previously described for discontinuous crystallization. The processing (separation and washing of the crystallized aminobenzoic acid) can also be carried out as previously described.

[0101] The formic acid required for the precipitation of the aminobenzoic acid can, in principle, originate from any source, unless it is obtained by reduction of carbon dioxide, in particular chemical or electrochemical reduction, as described in more detail below for a preferred embodiment.

[0102] INTRODUCING THE MOTHER LIQUOR CONTAINING FORMIATE ANIONS INTO THE FERMENTATION (STEP (D))

[0103] The mother liquor containing formate anions obtained in step (C) is introduced into the fermentation according to step (A1) in step (D). The aim is to select the proportion of mother liquor introduced into the fermentation as large as possible in order to be able to exploit the advantages of the invention to the maximum; however, it may be necessary to exclude a smaller portion of the mother liquor from the fermentation in order to avoid an accumulation of impurities in the process (so-called purge of a portion of the mother liquor for the purpose of removing impurities). Such a purge can be carried out periodically or at intervals and, if carried out, preferably comprises 0.5% to 45.0%, particularly preferably 1.0% to 25%, very particularly preferably 1.5% to 5.0% of the amount of mother liquor obtained in step (C).The remainder, i.e. preferably 55.0% to 99.5%, particularly preferably 75.0% to 99.0%, very particularly preferably 95.0% to 98.5%, of the mother liquor obtained in step (C) is then fed into the fermentation according to step (A1).

[0104] The discharged portion of mother liquor is depleted of aminobenzoic acid and fed to a wastewater treatment plant. The depletion of aminobenzoic acid can be carried out by processes known per se in the prior 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, preferably 9 to 11, carbon atoms, in particular 1-decanol. The extraction is preferably carried out as described in WO 2023 / 117756 A1 on page 19, line 15 to line 33. The dissolved aminobenzoic acid can be obtained by evaporating the extractant or by back-extraction as described in WO 2023 / 117756 A1 on page 20, line 1 to page 21, line 8.Activated carbon or a polymeric adsorbent, particularly based on polystyrene and / or polydivinylbenzene, is preferred as the adsorbent. Typical pore sizes range from 1.5 to 65 nm, e.g., 4.5 to 10 nm. Adsorbent types with micro- and macropores are also available. Commercially available products are available under the brand names 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 (and other adsorbents such as activated carbon) are suitable for both acidic (as described in WO 2018 / 114841 Al) and basic (as described in WO 2015 / 124687 Al) desorption. If desorption is performed in acidic conditions, it is recommended to perform an additional regeneration of the adsorbent with base from time to time (e.g., after five acidic desorptions).In this way, the adsorber bed is freed of organic contaminants. Regardless of the type of desorption, the desorbate is preferably fed into the crystallization process according to step (B).

[0105] Regardless of its size, the portion of the mother liquor obtained in step (C) fed into the fermentation according to step (A1) is preferably previously mixed with nutrients such as the aforementioned carbon and nitrogen sources or salts such as potassium phosphate, calcium chloride, or trace elements such as iron sulfate, manganese sulfate, copper sulfate, zinc sulfate, or nickel chloride. In addition, an agent for reducing foam in the fermentation, such as polypropylene glycol, can be added. It is possible to increase the pH of the mother liquor containing formate anions to a pH of > 5.5, in particular to 5.6 to 11, by adding a base (e.g., by adding aqueous or gaseous ammonia) before it is introduced into the fermentation according to step (A1). As already mentioned, however, this is surprisingly not absolutely necessary to maintain a constant pH during the fermentation.It is therefore preferred to pass the mother liquor containing formate anions into the fermentation according to step (Al) without changing the pH.

[0106] Particularly if pH adjustment is omitted, the mother liquor containing formate anions should be added slowly to the fermentation according to step (A1) to prevent formate from being added significantly faster than it is degraded (and consequently causing the pH to drop too much). It is therefore preferable to adjust the rate of addition of the mother liquor so that the pH of the fermentation broth does not change too much upon addition of the mother liquor.

[0107] The introduction of the formate anion-containing mother liquor into the fermentation according to step (A1) can be carried out by feeding the mother liquor into the same fermentation reactor from which the aminobenzoic acid precipitated in step (C) (more precisely: the aminobenzoate anions converted into aminobenzoic acid in step (B)) originally originated. It is also possible to operate several fermentation reactors in series and feed the resulting mother liquor to the downstream fermentation reactor. To start up the process, when no formate anion-containing mother liquor is yet available, the pH of the fermentation can be regulated by adding a base (e.g., by adding aqueous or gaseous ammonia, aqueous potassium hydroxide, or aqueous sodium hydroxide). The pH is preferably regulated during start-up by adding aqueous potassium hydroxide or aqueous sodium hydroxide.

[0108] In a batch process, the mother liquor containing formate anions obtained in one fermentation cycle can also be fed to the subsequent fermentation cycle. It makes no difference whether all fermentation cycles are carried out in the same fermentation reactor or in different fermentation reactors. Naturally, no mother liquor is available in the first fermentation cycle. This first fermentation reactor is then operated with pH controlled by adding base (e.g., aqueous or gaseous ammonia, aqueous potassium hydroxide, or aqueous sodium hydroxide).

[0109] As already mentioned, it was not expected that the introduction of the formate anion-containing mother liquor into the fermentation would be successful without a significant accumulation of formate anions in the fermentation broth. Rather, the formate anions are oxidized to carbon dioxide, incorporated into the target product of the fermentation, the aminobenzoate anions, and / or metabolized to form biomass. If necessary, formate degradation during the fermentation can be enhanced by the addition of formate-degrading enzymes. Examples of suitable enzymes are formate oxidases (EC 1.2.3.1) and formate dehydrogenases (EC 1.17.1).

[0110] USE OF AMINOBENZOIC ACID IN THE PRODUCTION OF OTHER VALUED PRODUCTS

[0111] The aminobenzoic acid obtained in step (C) is suitable, optionally after further purification by known methods (e.g. recrystallization), for all applications of aminobenzoic acid known in the prior art.

[0112] For example, ortho-aminobenzoic acid (anthranilic acid) plays an important role as a starting material for the synthesis of anthranilic acid esters, which are important fragrances, and indigo, as well as for pharmaceuticals and pesticides (acaricides). Anthranilic acid produced according to the invention is suitable for all of these purposes.

[0113] The use of aminobenzoic acid prepared according to the invention is preferred for the production of polymeric compounds. Here, the process according to the invention can make a valuable contribution to the more sustainable production of plastics, which are often required in large quantities. For example, anthranilic acid can be converted to a poly(anthranilic amide) as described in WO 2022 / 008449 A1 (after conversion to isatoic anhydride) or WO 2022 / 008450 A1 (after conversion to an anthranilic acid ester). Anthranilic acid obtained according to the invention can also be converted to an anthranilic acid derivative selected from anthranilic acid halide, isatoic anhydride, or a mixture thereof as described in WO 2022 / 122906 A1 and then reacted with a polyol to form a polyamine, which in turn is suitable for phosgenation to the corresponding polyisocyanate.

[0114] Aminobenzoic acid obtained according to the invention can also be decarboxylated to aniline, which is an important raw material, particularly in the polyurethane industry. As described in more detail below, the use of the aminobenzoic acid produced according to the invention for aniline production opens up the possibility of exploiting synergies with the production of aminobenzoic acid by fermentation and crystallization and is therefore particularly preferred.

[0115] The aniline obtained in this way can, in turn, be used for all known applications. In particular, the (acid-catalyzed) reaction with formaldehyde to form methylenediphenylenediamine and polymethylenepolyphenylenepolyamine, its phosgenation to form methylenediphenylene diisocyanate and / or polymethylenepolyphenylene polyisocyanate, and subsequent reaction with polyols to form polyurethanes should be mentioned. Of course, aniline obtained by decarboxylation of aminobenzoic acid prepared according to the invention can also be used for other applications, such as the production of azo compounds. One example is the production of methyl red, which is accessible by diazotization of the amino group of anthranilic acid with sodium nitrite and hydrochloric acid, followed by azo coupling with N,N-dimethylaniline.

[0116] DECARBOXYLATION OF AMINOBENZOIC ACID AND USE OF THE FORMED CARBON DIOXIDE FOR THE PRODUCTION OF FORMIC ACID

[0117] As already mentioned, the decarboxylation of aminobenzoic acid to aniline is a particularly preferred further embodiment of the process. The carbon dioxide released can be converted to formic acid using known methods (in particular chemical or electrochemical reduction), which can then be used in step (B). This provides, apart from possible losses, exactly the amount of formic acid required stoichiometrically for the conversion of aminobenzoate anions to aminobenzoic acid.

[0118] • 1 mole of protons is required per mole of aminobenzoic acid produced from aminobenzoate anions,

[0119] • During decarboxylation to aniline, 1 mole of carbon dioxide is produced per mole of aminobenzoic acid converted,

[0120] • To produce one mole of formic acid by reduction, one mole of carbon dioxide is required.

[0121] This embodiment therefore ideally allows (apart from yield losses, for example) the stoichiometrically required portion of the total formic acid required to be provided through a recycle process. Furthermore, the carbon dioxide released in stoichiometric amounts during decarboxylation is chemically rebound (and not released into the atmosphere), which is advantageous in itself.

[0122] Decarboxylation

[0123] Decarboxylation can be carried out as is generally known in the art. A catalyst may, but is not required.

[0124] Suitable catalysts include, for example, 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 hydroxyapatites and hydrotalcites; and polymeric acids such as ion exchange resins (preferably Amberlyst). Particularly preferred catalysts are those described in WO 2022 / 253890 A1. The catalysts disclosed therein are characterized by a high aluminum oxide mass fraction (at least 40%). The aluminum oxide is preferably γ-Al2O3 or p-Al2O3, especially when no other metal oxides are present besides aluminum oxide. In addition to aluminum oxide, other metal oxides may in principle also be present, in particular magnesium oxide (MgO) in a mass fraction 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 fraction of 1.0% to 30.0%, preferably 2.0% to 20.0%, particularly preferably 2.0% to 10.0%, based on its total mass.

[0125] As for the reaction conditions, the decarboxylation can be carried out over a wide range of temperature and pressure. A suitable reaction temperature is preferably in the range of 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 most preferably 1.0 bar to 60 bar.

[0126] The decarboxylation can also be carried out in the presence of aniline, i.e., the aminobenzoic acid is dissolved in aniline. Since aniline exerts a catalytic effect with respect to the decarboxylation of aminobenzoic acid, thus accelerating its own formation (autocatalytic effect), the use of a catalyst external to the system is not absolutely necessary in this variant; see also WO 2020 / 020919 A1, which describes such a process. In contrast to the procedure disclosed in WO 2020 / 020919 A1, purified aniline can also be used as a solvent for the aminobenzoic acid. Furthermore, in contrast to the procedure disclosed in WO 2020 / 020919 A1, external catalysts can also be used.When the reaction is carried out discontinuously, a mass fraction of aniline, based on the total mass of aniline and aminobenzoic acid, of 0.1% to 90%, preferably 1.0% to 70%, particularly preferably 5.0% to 50%, is preferably set before the start of the decarboxylation. When the reaction is carried out continuously, a mass fraction of aniline, based on the total mass of aniline and aminobenzoic acid, of 0.1% to 90%, preferably 1.0% to 70%, particularly preferably 5.0% to 50%, is preferably set during the decarboxylation.

[0127] In addition to aniline, other solvents or diluents can of course also be used, especially water. Furthermore, organic, polar, or protic solvents are preferably suitable, such as halogenated aliphatic or aromatic hydrocarbons, linear or cyclic ethers, linear or cyclic esters, linear or cyclic amides, alcohols, ketones, nitriles, phenol derivatives, benzanilides, sulfonamides, or sulfolane, which preferably have a boiling point that, under the selected conditions, is higher than the selected reaction temperature and, at this temperature, preferably forms a homogeneous reaction mixture with the reaction components.

[0128] Regarding the reaction procedure, both the gas phase and the liquid phase are suitable. The reaction can be carried out continuously (preferred) or discontinuously. Preferred procedures involve the decarboxylation of the aminobenzoic acid.

[0129] • in the liquid or gas phase in a reactor, in particular in a tubular reactor, with an integrated fixed bed of the catalyst (including a bed of the catalyst as a shaped body (extrudates) or a design of the catalyst as a monolithic structure),

[0130] • in the liquid or – preferably – gas phase in a fluidized bed reactor or

[0131] • in the liquid phase in a stirred tank containing a suspension (slurry) of the catalyst.

[0132] A tubular reactor is understood here as a tubular reactor through which the reacting reaction mixture flows during operation in a continuous reaction (which is preferred). Tubular reactors with small length-to-diameter ratios are also referred to as tower reactors; these are also encompassed by the underlying understanding of the term "tubular reactor," as are special designs of tubular reactors such as bubble column reactors.

[0133] The use of catalyst moldings (extrudates) or monolithic catalyst structures allows easy reuse of the catalyst after decarboxylation.

[0134] The catalyst remaining after decarboxylation is preferably regenerated before reuse. This can be done by washing the catalyst with organic solvents or aqueous solutions and / or burning it out at elevated temperatures in the presence of O2 to remove organic deposits.

[0135] The aniline formed can be isolated and purified by standard techniques, particularly distillation, and used as described above.

[0136] Chemical or electrochemical reduction of carbon dioxide

[0137] As explained above, the carbon dioxide produced during decarboxylation is preferentially converted into formic acid. Carbon dioxide from decarboxylation is particularly suitable for this purpose because it contains only trace amounts of aniline or anthranilic acid as impurities, which can be easily separated.

[0138] Another system-immanent source of carbon dioxide for the purpose of conversion to formic acid can be carbon dioxide formed during the fermentation according to step (A1) (particularly by oxidation of the formate anions). However, this source of carbon dioxide is less preferred, since the carbon dioxide obtained in this way is obtained in diluted form. Of course, carbon dioxide from any external source can also be used, provided it is obtained in sufficient purity or can be purified with reasonable effort.

[0139] The conversion of carbon dioxide to formic acid takes place using processes that are known per se and are therefore only briefly described below.

[0140] The chemical reduction of carbon dioxide is carried out by hydrogenation with hydrogen using suitable catalysts, particularly ruthenium and iridium catalysts with nitrogen- and / or phosphorus-containing ligands. Suitable processes are described, for example, in Chem. Rev. 2015, 115, 12936-12973 by W.-H. Wang, Y. Himeda, J.T. Muckerman, GF Manbeck, and E. Fujita ("CO Hydrogenation to Formate and Methanol as an Alternative to Photo- and Electrochemical CO2 Reduction") [1]. For the presently relevant purpose of using formic acid in the crystallization of aminobenzoic acid, a process should be chosen that actually yields formic acid as the final product (as described, for example, in Scheme 2 of [1]) and not a formate salt. (In principle, a formate salt can, of course, easily be converted into formic acid; however, such an additional step would significantly impair the economic viability of the process.)

[0141] A process using ruthenium catalysts, such as [Ru(H)2(PnBu3)4], using amines, such as trihexylamine, and polar (hydrogen-bonding-capable) solvents such as diols (particularly 2-methyl-1,3-propanediol, 1,3-propanediol, 1,2-propanediol, or ethanediol) was described by T. Schaub and R.A. Paciello in Angew. Chem. 2011, 123, 7416-7420 {"A process for the production of formic acid by CCH hydrogenation: thermodynamics and the role of CO") [3]. A multiphase liquid-liquid process concept is disclosed, allowing the recovery of the amine, the polar solvent, and the catalyst, with formic acid being separated from the system by distillation.

[0142] The electrochemical reduction of carbon dioxide occurs by acid electrolysis, i.e., at pH values ​​below the pKa of formic acid (3.77), particularly at pH 2.00 to 3.75. Suitable electrolysis cells and operating methods for this purpose are described by M. Oßkopp, A. Löwe, CMS Lobo, S. Baranyai, T. Khoza, M. Auinger, and E. Klemm in Journal of CO2 Utilization 2022, 56, 101823 ("Producing formic acid at low pH values ​​by electrochemical CO2 reduction") [5].

[0143] The process described above makes it possible to reduce the salt load in the wastewater and improve the yield of aminobenzoic acid, since at least the majority of the mother liquor is returned to the fermentation, thus preventing the dissolved aminobenzoic acid from being lost. The following examples illustrate the process in more detail. Examples:

[0144] Example 1 (Precipitation of aminobenzoic acid with formic acid)

[0145] A fermentation broth containing aminobenzoate anions at a concentration of 55.9 g / L (calculated as aminobenzoic acid) with a pH of 7.8 was adjusted to a pH of 5.7 by adding formic acid (>98%) over 30 minutes. Seed crystals of aminobenzoic acid were then added in an amount corresponding to 1% of the calculated mass of aminobenzoic acid present in the fermentation broth. After a waiting time of 30 minutes, the pH was reduced to pH 3.7 over 60 minutes by adding additional formic acid. The mixture was stirred at 200 rpm throughout. The resulting suspension of aminobenzoic acid in mother liquor was filtered through a suction filter under a diaphragm pump vacuum. The moist filter cake was washed with hydrochloric acid (pH 3.5) and dried overnight at 60 °C under vacuum at 30 mbar. The yield of isolated and dried aminobenzoic acid was 79 wt. %.

[0146] Example 2 (Formate degradation with C. qlutamicum]

[0147] To prepare the cells, C. glutamicum ATCC 13032 was inoculated in Brain-Heart Infusion Medium (ready-made preparation from Oxoid) in an Erlenmeyer flask with an OD 500 of 0.005 and incubated for 24 h at 30 °C and 200 rpm. The cells were then harvested by centrifugation at 8000 rpm and 4 °C for 10 min. The cell pellet was stored at -20 °C until use.

[0148] The cell pellet was thawed and then resuspended in Mcllvaine's buffer (citrate-phosphate buffer, prepared as described in "A BUFFER SOLUTION FOR COLORIMETRIC COMPARISON", T.C. Mcllvaine, Journal of Biological Chemistry, 1921, 49(1), 183-186 (doi:10.1016 / S0021-9258(18)86000-8) [4]). Reactions to monitor formate degradation were carried out at a normalized OD500 of 5.0 at various pH values ​​in Mcllvaine's buffer in overnight culture tubes with a 2 mL reaction volume and initiated by the addition of sodium formate. The tubes were incubated at 30 °C and 200 rpm. Samples for formate quantification were taken after 0, 2, 4, 6, and 24 h and incubated at 95 °C for 5 min to inactivate. Cell components were then separated by centrifugation at 13,000 rpm for 5 min. The supernatant was stored at 4 °C until formate quantification. Formate was quantified photometrically via the enzymatic formation of NADH.The samples were diluted in the reaction buffer (50 mM potassium phosphate buffer, pH 7.6) depending on the expected formate concentration. After addition of 1 U / mL formate dehydrogenase from C. boidinii and 1 mM NAD. + The reaction was incubated at 37 °C for 3 h. Subsequently, the absorbance at 340 nm was determined, and the formate concentration was determined using a calibration curve. The time course of the formate concentration is shown in Table 1. The fastest formate degradation was observed at pH 6.0.

[0149] Table 1: Time course of formate concentration at different pH values. mM = mmol / L

[0150] Example 3 (Fermentation of C. glutamicum with addition of formate-containing mother liquor)

[0151] Addition of sodium formate-containing mother liquor to the cultivation of C. glutamicum

[0152] The microbial strain described below was used:

[0153] Based on the bacterium Corynebacterium glutamicum ATCC13032, a microbial strain was induced to produce anthranilic acid through targeted chromosomal modifications. All genetic modifications, i.e., chromosomal deletions and gene integration, were performed by double homologous recombination using corresponding pK19mohsacß 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(l):69-73. doi: 10.1016 / 0378-1119(94)90324-7) [6].

[0154] The activity of the anthranilate phosphoribosyltransferase TrpD was reduced by first deleting the native trpD-A\\e\ (SEQ ID NO. 1) and replacing it with an allele (called trpD5) with GTG instead of ATG start codon and ribosome binding site with reduced distance to the start codon (SEQ ID NO. 2).

[0155] Furthermore, phosphoenolpyruvate carboxylase was inactivated by largely deleting the corresponding gene as described in (SEQ ID NO. 3).

[0156] To enhance the aromatic biosynthesis pathway, an artificial, polycistronic P tU f- aroLAC operon (SEQ ID NO. 4), consisting of the genes aroL (b0388), encoding a shikimate kinase from Escherichia coli, aroA (cg0873), encoding the 3-phosphoshikimate-l-carboxyvinyltransferase from C. glutamicum, and aroC (cgl829), encoding the chorismate synthase from C. glutamicum, was integrated downstream of cg2563 under the control of the constitutive promoter of the elongation factor Tuf. In addition, the construct P t uf-oroG new (SEQ ID NO. 5), which encodes a feedback-resistant variant of DAHP synthase from E. coli under control of P tU f- Pro motors encoded, integrated downstream of cg3132 into the genome of the strain.

[0157] For pre-cultivation of the C. glutamicum strain, it was first transferred from a cryo-culture into sterile SY medium (10 g / L yeast extract, 16 g / L soy peptone, 5 g / L NaCl, 16 g / L glucose) and incubated in 1 L Erlenmeyer flasks with 50 mL liquid culture at 30 °C and 200 rpm until an OD600 of approximately 30 was reached. 5 mL / flask of this first preculture was transferred to sterile modified CGXII medium (5 g / L yeast extract, 10 g / L (NH^2SC, 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 incubated under otherwise identical conditions until an OD600 of approximately 40 is reached.The final cultivation stage ("seed"), like the main cultivation, was carried out in stirred bioreactors (BioBlock, Eppendorf) under aerobic conditions (dissolved oxygen concentration > 30%). 80 mL of the second preculture were dissolved in 520 mL seed 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 MgSO4x 7 H2O, 0.07 g / L CaCl2x 2 H2O, 4.17 g / L PPG 2000, 167 mg / L MnSO4x H2O, 167 mg / L FeSO4x 7 H2O, 16.7 mg / L ZnSO4x 7 H2O, 3.34 mg / L CuSO4x 5 H2O, 0.334 mg / L iCl2x 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 with the addition of a glucose-tryptophan mixture (480 g / L glucose, 1.6 g / L tryptophan), as well as a mixture of NaOH and NH3 solution (1.55 mol / L NaOH, 8 mass% NH3) for pH control. After reaching an OD of approx.200, 80 mL of the seed culture were transferred into each main cultivation mixture 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 main fermentation was carried out at 33 °C and pH 7.0 with the addition of a 600 g / L glucose solution, a 20% NHs solution for pH control and sodium formate-containing mother liquor from Example 1 or synthetic solution.

[0158] The mother liquor was adjusted to pH 7.0 with NaOH solution before use and sterile 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 an HPLC system 126011 Infinity, Agilent Technologies).

[0159] As an alternative to real mother liquor, a synthetic solution containing 35.7 g / L sodium formate (24.2 g / L formate) and 8 g / L anthranilic acid was prepared, adjusted to pH 7.0 with NaOH solution, and sterile-filtered. Different addition rates were selected for the cultivations with the synthetic solution. In Tables 2 to 6, these are simply labeled "low" and "increased." The formate addition at the "increased" addition rate of the synthetic solution corresponded to the addition rate when dosing the mother liquor. The formate addition at the "low" addition rate of the synthetic solution was 20% lower. As shown in Table 2, no cultivation showed a significant increase in the formate concentration. Regardless of different addition rates and formate concentrations of the added solutions, > 99% of the added formate was degraded by the organisms in every case. As shown in Table 3, this allowed for approximately48 to 139% of the Na required for anthranilic acid production. + -Counterions via the supplied

[0160] (synthetic or real) mother liquor. The anthranilic acid recycled via the mother liquor is not included in the produced anthranilic acid, as this was already neutralized during the preparation of the mother liquor. Depending on the batch, the recycled anthranilic acid accounted for approximately 5.4 to 9.8% of the final anthranilic acid.

[0161] Table 2: Maximum formate concentration and proportion of formate consumed

[0162] [a] The experiments were each carried out twice under the same conditions in separate reaction apparatus; in the following, such duplicated experiments are referred to as "(1)" and "(2)".

[0163] Table 3: Ratio of sodium counterions added via the synthetic mother liquor to the anthranilic acid produced Tables 4 to 6 show the absolute amounts of microbial dry biomass, anthranilic acid, and degraded formate during the various cultivations. Due to a temporary glucose limitation in the "Mother Liquor (1)" preparation after approximately 20 hours, this preparation exhibits slightly lower amounts of dry biomass and anthranilic acid. The fact that the amount of degraded formate, as shown in Table 6, is nevertheless higher than the amount degraded formate in "Mother Liquor (2)" indicates that the cells were not yet limited in their capacity to metabolize formate. Overall, the results demonstrate that growth and product formation are possible with simultaneous recycling of formate-containing mother liquor.

[0164] Table 4: Absolute amount of dry microbial biomass during cultivation with supplementation of formate-containing mother liquor / solution

[0165] Table 5: Absolute amount of anthranilic acid during cultivation with supplementation of formate-containing mother liquor / solution Table 6: Absolute amount of formate degraded during cultivation with supplementation of formate-containing mother liquor / solution

[0166] Example 4 (Formate degradation with different microorganisms) Cultivation of different microorganisms for formate degradation

[0167] The cultivation conditions are summarized in Table 7. Precultures were seeded with cell material from a cryoculture or solid medium and incubated overnight. The main culture was seeded at 1 / 200 and cultivated for 48 h before the cells were harvested by centrifugation.

[0168] Table 7: Cultivation conditions Nutrient Broth Medium

[0169] For one liter of liquid medium, 25 g of ready-to-use powder (Invitrogen / Thermo Fisher Scientific) was dissolved in one liter of deionized water. The liquid medium contained:

[0170] • 3 g / L yeast extract

[0171] • 1 g / L glucose

[0172] • 6 g / L NaCl

[0173] • 15 g / L peptone

[0174] The solution was autoclaved and then stored at room temperature.

[0175] Yeast Malt Medium

[0176] For one liter of liquid medium, 21 g of ready-to-use powder (NutriSelect) was dissolved in one liter of deionized water. The liquid medium contained:

[0177] • 10 g / L glucose

[0178] • 3 g / L malt extract

[0179] • 5 g / L peptone

[0180] • 3 g / L yeast extract

[0181] The solution was autoclaved and then stored at room temperature.

[0182] Yeast Peptone Dextrose (YPD) Medium

[0183] For one liter of the liquid medium, a 100 g / L (5x) glucose monohydrate solution was prepared (Solution 1). As a second solution, 20 g of peptone and 10 g of yeast extract were dissolved in deionized water and made up to 0.8 L. The solutions were autoclaved separately and then stored at room temperature. For the final medium, 0.8 L of Solution 2 was made up to 0.2 L of Solution 1. The liquid medium contained:

[0184] • 20 g / L glucose monohydrate

[0185] • 20 g / L peptone

[0186] • 10 g / L yeast extract

[0187] An adapted YPD medium was used to cultivate K. pastoris. 0.5% (v / v) methanol was used as the carbon source instead of glucose monohydrate. Additionally, 10% (v / v) of a 100 g / L (NH^2SC) solution and 0.2% (v / v) of a 200 mg / L biotin solution were used. These solutions were prepared with deionized water and sterile filtered. The biotin solution was stored at 4 °C and the (NH^2SC) solution at room temperature. To obtain the same concentrations as in the original YPD medium, 9.3% (v / v) of deionized and autoclaved water was added.

[0188] Formate degradation was carried out in 15 mL culture tubes containing 2 mL of reaction solution in McIllvaine buffer at 20 °C and 200 rpm. pH 5 was used for yeast and pH 6 for bacteria. The cells were used in the reaction with a final OD 500 of 5. For this purpose, after cultivation, the equivalent amount of cell culture was centrifuged for 10 min at 9000 rpm before the cell pellet was resuspended in the reaction buffer. The reaction was initiated by the addition of 7.5 mM sodium formate. Samples were taken during the reaction and used for formate quantification.

[0189] MTP-based FDH assay for the photometric quantification of formate

[0190] To quantify formate in samples, the robust FDH assay based on the formate dehydrogenase CbFDH was used. The required solutions, including volumes and concentrations, are listed in Table 8. Either sodium formate solution was used for the standard line or sample. The dilutions for determining the standard line were prepared with potassium phosphate buffer. From the NAD solutions, + A master mix was prepared from the CbFDH, and buffer, corresponding to the volumes used in the assay, which was pipetted into the individual wells to start the assay. The sample or formate solution was added to the wells. The assay was performed at 37 °C for 3 h in a photometer. The wells were measured at 340 nm.

[0191] Table 8: Solutions used in the CbFDH assay as well as volumes and concentrations, V ges = 200 pL

[0192] All strains were able to degrade significant amounts of formate. While no formate was detectable in K. pastoris after 4.1 h, 1.4 mM was still detectable in P. putida and 6.2 mM in B. o. polymorpha. After 24.5 h, no more formate could be detected in any of the strains.

Claims

Patent claims:

1. A process comprising the preparation of aminobenzoic acid, the process comprising the following steps: (A) Providing a fermentation broth containing aminobenzoate anions, comprising: (A1) fermenting a fermentable carbon-containing compound and a nitrogen-containing compound in the presence of microorganisms selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans or a mixture of two or more of the aforementioned microorganisms at a pH of greater than 5.5 and (A.2) separating the microorganisms; (B) adjusting the pH of the fermentation broth containing aminobenzoate anions to 3.0 to 5.5 by adding formic acid, whereby aminobenzoic acid precipitates; (C) separating aminobenzoic acid precipitated in step (B) to obtain a mother liquor containing formate anions and formic acid; and (D) introducing 55.0% to 100% of the total mother liquor obtained in step (C) into the fermentation according to step (A1) with depletion of the formate anions and formic acid by (i) an added enzyme preparation, and / or (ii) the microorganisms used in step (A) and / or (iii) added further microorganisms which are different from the microorganisms used in step (A).

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

3. A process according to claim 1 or 2, wherein the nitrogen-containing compound is selected from ammonia gas, ammonia water, an ammonium salt, soy protein, urea or a mixture of two or more of the aforementioned nitrogen-containing compounds.

4. A process according to any one of the preceding claims, wherein the fermentable carbon-containing compound is selected from starch hydrolysate, an alkali metal or ammonium formate, sugar cane juice, sugar beet juice, hydrolysates from lignocellulose-containing raw materials or a mixture of two or more of the aforementioned carbon-containing compounds.

5. Process according to one of the preceding claims, in which in step (A1) a pH of 5.6 to 11 is maintained.

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

7. A process according to any one of the preceding claims, wherein ortho-aminobenzoic acid or para-aminobenzoic acid is produced.

8. A process according to any one of the preceding claims, wherein the aminobenzoic acid from step (C) is converted to aniline with elimination of carbon dioxide.

9. The process according to claim 8, wherein the carbon dioxide formed in the reaction to aniline is converted to formic acid and the formic acid thus obtained is used in step (B).

10. A process according to any one of the preceding claims, wherein carbon dioxide is formed in the fermentation according to step (A1) and this carbon dioxide is converted to formic acid and the formic acid thus obtained is used in step (B).

11. A process according to claim 9 or 10, wherein the conversion to formic acid is carried out by hydrogenation or acid electrolysis of the carbon dioxide.

12. A process according to any one of the preceding claims, wherein in step (D) the first part of the mother liquor is admixed with a further carbon-containing compound, a further nitrogen-containing compound, a compound for reducing foam formation in the fermentation, an inorganic salt, a trace element or a mixture of two or more of the aforementioned compounds before it is introduced into the fermentation.

13. A process according to any one of the preceding claims, wherein in step (D) less than 100% of the total mother liquor obtained in step (C) is introduced into the fermentation according to step (A1), wherein the part of the mother liquor not introduced into the fermentation is fed to a wastewater treatment plant after being depleted of aminobenzoic acid.

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

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

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