WO2025132393A1 - Production of aminobenzoic acid - Google Patents

Abstract

The invention relates to a method involving the production of aminobenzoic acid, said method having the steps of: (A) fermenting a fermentable carbon-containing compound and a nitrogen-containing compound in the presence of microorganisms to obtain a fermentation broth containing aminobenzoate anions and/or aminobenzoic acid; (B) recovering aminobenzoic acid from the fermentation broth; (C) converting carbon dioxide into a reduction product selected from formic acid, ammonium formate, an alkali metal salt of formic acid, acetic acid, ammonium acetate, or an alkali metal salt of acetic acid, and (D) adding the reduction product from step (C) to the fermentation process of step (A).

Description

PRODUCTION OF AMINOBENZOIC ACID

The present invention relates to a process comprising the production of aminobenzoic acid, the process comprising: (A) fermenting a fermentable carbon-containing compound and a nitrogen-containing compound in the presence of microorganisms to obtain a fermentation broth containing aminobenzoate anions and/or aminobenzoic acid, (B) recovering aminobenzoic acid from the fermentation broth, (C) converting carbon dioxide to a reduction product selected from formic acid, ammonium formate, an alkali metal salt of formic acid, acetic acid, ammonium acetate or an alkali metal salt of acetic acid, and (D) adding the reduction product from step (C) to the fermentation of step (A).

The production of organic acids through fermentative 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, as well as the corresponding polyisocyanates. For example, according to WO 2022/008450 Al, anthranilic acid can be converted into an anthranilic acid ester, which can then be catalytically converted into poly(anthranilamide) by polycondensation with elimination of the alcohol underlying the anthranilic acid ester.

The fermentative production of aminobenzoic acid is generally known in the prior 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.

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 in order 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 describe the use of bacteria from the Corynebacteria or Pseudomonads families as possible recombinant microorganisms. A more recent application (WO 2017/102853 A1) describes the use of yeasts.

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.

Fermentation processes regularly produce carbon dioxide-containing offgases. This applies to both aerobic and anaerobic processes, such as bioethanol production. The state of the art generally does not address the further use of such carbon dioxide. EP 3 715464 B1 describes a process in which a microorganism is cultivated in a bioreactor, CO2 is captured from the bioreactor, reduced to an organic starting material, and the organic starting material is at least partially fed into the bioreactor.

Carbon dioxide can also be produced in a reaction downstream of the fermentation, such as the production of aniline from aminobenzoic acid described above.

Ralf Takors et al. describe in Microbial Biotechnology 2018, 11 (4), 606-625 ("Using gas mixtures of CO, CO2, and H as microbial substrates: the do's and don'ts of successful technology transfer from laboratory to production scale") [1] the use of gas mixtures of CO, CO2, and H2 for gas fermentations (see Fig. 2 of [1]). Markus Stöckl et al. describe in ChemSusChem 2020, 13, 4086-4093 ("From CO2 to Bioplastic - Coupling the Electrochemical CO2 Reduction with a Microbial Product Generation by Drop-in Electrolysis") [2] the electrochemical reduction of CO2 to formate and the use of formate as the sole substrate in the production of polyhydroxybutyrate by the microorganism Cupriavidus necator.

The biotechnological production of aminobenzoic acid can, in itself, make a valuable contribution to resource conservation and emissions reduction. A process that would enable the carbon dioxide produced during fermentation or otherwise generated to be reused in the fermentation itself, thus making it even more sustainable, has not yet been described. There was therefore a need for further improvements in the field of fermentative production of aminobenzoic acid. In particular, it would be desirable to be able to utilize carbon dioxide (i) formed during fermentation, (ii) formed in subsequent steps, and/or (iii) from external sources.

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

(A) (preferably aerobic) fermentation of a fermentable carbon-containing compound and a nitrogen-containing compound in the presence of microorganisms to obtain a fermentation broth containing aminobenzoate anions and/or aminobenzoic acid;

(B) recovering aminobenzoic acid from the fermentation broth;

(C) converting carbon dioxide to a reduction product selected from formic acid, ammonium formate (the ammonium salt [NH salt] of formic acid), an alkali metal salt (especially the sodium or potassium salt) of formic acid, acetic acid, ammonium acetate (the ammonium salt [NH salt] of acetic acid) or an alkali metal salt (especially the sodium or potassium salt) of acetic acid, with acetic acid, ammonium acetate or an alkali metal salt of acetic acid being preferred and ammonium acetate or an alkali metal salt of acetic acid being particularly preferred; and

(D) Addition of the reduction product from step (C) to the fermentation of step (A).

Completely surprisingly, it was found that formic acid, acetic acid or salts thereof, preferably acetic acid and its salts, particularly preferably salts of acetic acid, produced by reduction of carbon dioxide are suitable for being fed into a fermentation for the production of aminobenzoic acid and for being utilized there.

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

A fermentable carbon-containing compound in the sense of the present invention is any organic compound or mixture of organic compounds which can be used by the microorganisms employed to produce aminobenzoic acid. The production of aminobenzoic acid can take place in the presence (aerobic) or absence (anaerobic), preferably in the presence of oxygen (aerobic), especially in the form of an oxygen-containing gas such as air.

A reduction product of carbon dioxide is understood to be a reaction product of carbon dioxide in which the oxidation number of the carbon atom of the product corresponding to the carbon atom of carbon dioxide is reduced compared to the carbon atom. According to the invention, the reduction products are selected from formic acid, ammonium formate, an alkali metal salt of formic acid, acetic acid, ammonium acetate, or an alkali metal salt of acetic acid.

For the purposes of the present invention, an external source of carbon dioxide is understood to mean a source that is different from the fermentative production of aminobenzoic acid or the production of a direct reaction product of aminobenzoic acid. A direct reaction product of aminobenzoic acid refers to a product that can be obtained directly, i.e., without intermediates, by reacting aminobenzoic acid. An immediate reaction product in this sense is aniline formed by decarboxylation.

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

In a first embodiment of the invention, which can be combined with all other embodiments, carbon dioxide formed in the fermentation of step (A) is not converted into the reduction product in step (C).

In a second 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 produced in the process according to the invention.

In a third embodiment of the invention, which can be combined with all other embodiments except those limited to the formation of meta- or ortho-aminobenzoic acid, para-aminobenzoic acid is produced in the process according to the invention.

In a fourth embodiment of the invention, which is a particular embodiment of the second embodiment, the ortho-aminobenzoic acid is converted to a poly(anthranilamide).

In a fifth embodiment of the invention, which is a further particular embodiment of the second embodiment, the ortho-aminobenzoic acid is converted to a anthranilic acid derivative selected from an anthranilic acid halide, isatoic anhydride or a mixture thereof, and the anthranilic acid derivative is reacted with a polyol to form a polyamine.

In a sixth embodiment of the invention, which is a particular embodiment of the fifth embodiment, the polyamine is phosgenated to a polyisocyanate.

In a seventh 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 second embodiment, the process comprises a step (E) in which the aminobenzoic acid from step (B) is decarboxylated to aniline and carbon dioxide formed in the process is converted in step (C) to the reduction product.

In an eighth embodiment of the invention, which is a particular embodiment of the seventh embodiment, the aniline is reacted with formaldehyde to form methylenediphenylenediamine and polymethylenepolyphenylenepolyamine.

In a ninth embodiment of the invention, which is a particular embodiment of the eighth embodiment, the methylenediphenylenediamine and/or the polymethylenepolyphenylenepolyamine is phosgenated to methylenediphenylene diisocyanate and/or polymethylenepolyphenylene polyisocyanate.

In a tenth embodiment of the invention, which is a particular embodiment of the ninth embodiment, the methylenediphenylene diisocyanate and/or the polymethylenepolyphenylene polyisocyanate is reacted with a polyol to form a polyurethane.

In an eleventh embodiment of the invention, which is a further particular embodiment of the seventh embodiment, the aniline is converted to an azo compound.

In a twelfth embodiment of the invention, which can be combined with all other embodiments, in step (C) carbon dioxide from an external source is converted to the reduction product.

In a thirteenth embodiment of the invention, which is a particular embodiment of the twelfth embodiment, the carbon dioxide originates from an external source from a process for producing (i) bioethanol, (ii) cement, (iii) hydrogen, in particular for ammonia synthesis, or (iv) epoxides, or a biological degradation process (in particular in a sewage treatment plant or biogas plant) in a digester, or from a combustion of fuels, or a process for extracting carbon dioxide from air (so-called direct air capture) In a fourteenth embodiment of the invention, which can be combined with all other embodiments, step (C) comprises a reaction of the carbon dioxide with hydrogen or an electrolysis of the carbon dioxide.

In a fifteenth embodiment of the invention, which can be combined with all other embodiments except those which necessarily provide for a narrower selection of carbon-containing compounds, the fermentable carbon-containing compound is selected from starch hydrolysate, formic acid, an alkali metal salt of formic acid, ammonium formate, acetic acid, an alkali metal salt of acetic acid, ammonium acetate, 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.

In a sixteenth embodiment of the invention, which can be combined with all other embodiments, step (B) comprises crystallization and/or extraction.

In a seventeenth 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.

In an eighteenth embodiment of the invention, which can be combined with all other embodiments except those that exclude the microorganisms mentioned below, the microorganisms in step (A) are selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans, or a mixture of two or more of the aforementioned microorganisms. Corynebacterium glutamicum is preferred. Corynebacterium glutamicum ATCC13032 is particularly preferred.

In a nineteenth embodiment of the invention, which is a particular embodiment of the eighteenth embodiment, the reduction product in step (C) is selected from ammonium formate, an alkali metal salt of formic acid, ammonium acetate or an alkali metal salt of acetic acid.

In a twentieth embodiment of the invention, which is a particular embodiment of the eighteenth and nineteenth embodiments, step (A) is carried out in a pH range of 5.5 to 11, preferably 6.0 to 8.0, to obtain a fermentation broth containing aminobenzoate anions.

In a twenty-first embodiment of the invention, which is a particular embodiment of the eighteenth, nineteenth and twentieth embodiments, the fermentable carbon-containing compound is selected from starch hydrolysate, an alkali metal salt of Formic acid, ammonium formate, an alkali metal salt of acetic acid, ammonium acetate, 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.

In a twenty-second embodiment of the invention, which can be combined with all other embodiments except those which exclude the microorganisms mentioned below, the microorganisms in step (A) are selected from Ashbya gossypii, Pichia pastoris, Hansenula polymorpha, Kluyveromyces marxianus, Yarrowia lipolytica, Zygosaccharomyces bailii, Saccharomyces cerevisiae or a mixture of two or more of the aforementioned microorganisms.

In a twenty-third embodiment of the invention, which is a particular embodiment of the twenty-second embodiment, the reduction product is selected from formic acid or acetic acid.

In a twenty-fourth embodiment of the invention, which is a particular embodiment of the twenty-second and twenty-third embodiments, step (A) is carried out in a pH range of 3.0 to <5.5, preferably 3.5 to 5.0, to obtain a fermentation broth containing aminobenzoic acid and/or aminobenzoate anions.

In a twenty-fifth embodiment of the invention, which is a particular embodiment of the twenty-second, twenty-third and twenty-fourth embodiments, the fermentable carbon-containing compound is selected from starch hydrolysate, formic acid, acetic acid, 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.

In a twenty-sixth embodiment of the invention, which can be combined with all other embodiments except those which necessarily provide for a narrower selection of microorganisms, the microorganisms in step (A) are selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans, Ashbya gossypii, Pichia pastoris, Hansenula polymorpha, Kluyveromyces marxianus, Yarrowia lipolytica, Zygosaccharomyces bailii, Saccharomyces cerevisiae or a mixture of two or more of the aforementioned microorganisms.

The embodiments briefly described above and other possible configurations of the invention are explained in more detail below. All of the embodiments described above and the further configurations 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. FERMENTATION

Step (A) of the process according to the invention relates to the fermentation of a fermentable carbon-containing compound and a nitrogen-containing compound in the presence of microorganisms to obtain a fermentation broth containing aminobenzoate anions (H2NC6H4COO) and/or aminobenzoic acid (H2NC6H4COOH or HshFCet COCT). 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 (A) is preferably carried out such that the pH in the fermentation broth is in the range of 3.0 to 11. 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 excessively low pH values) or by adding an aqueous acid, in particular hydrochloric acid, sulfuric acid, or nitric acid (at excessively high pH values). Different pH ranges within the stated ranges may be particularly optimal for different microorganisms; this will be explained in more detail below.

Preferred microorganisms for carrying out step (A) are prokaryotes (such as, in particular, bacteria) or eukaryotes (such as, in particular, yeasts). Suitable microorganisms are, in particular, Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans, Ashbya gossypii, Pichia pastoris, Hansenula polymorpha, Kluyveromyces marxianus, Yarrowia lipolytica, Zygosaccharomyces bailii, and Saccharomyces cerevisiae. The use of mixtures of different microorganisms is possible, but the use of microorganisms of a single species is preferred.

In order to obtain such prokaryotes or eukaryotes, there are basically two ways available, which can also be combined in a preferred embodiment:

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

(ii) The subsequent reactions by which aminobenzoic acid is converted into further metabolites or products (e.g. tryptophan) can be reduced or eliminated, so that even the rate of aminobenzoic acid formation is sufficient to lead to an accumulation of aminobenzoic acid in the cell.

Methods for obtaining prokaryotic or eukaryotic organisms with the aforementioned properties are known in the art. Suitable prokaryotes or eukaryotes can be identified, for example, by screening for mutants that release aminobenzoic acid into the surrounding medium. However, the targeted modification of key enzymes by genetic engineering is preferred. Gene expression and enzyme activity can be enhanced, reduced, or even completely inhibited using conventional genetic engineering methods. This results in recombinant strains. A preferred embodiment for the particularly preferred ortho isomer is described below; transfer to the other isomers is within the scope of conventional expert knowledge:

Particularly preferably, the prokaryotes or eukaryotes 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.

In yeast, anthranilate phosphoribosyltransferase activity is genetically encoded by the native gene TRP4 (YDR354W). In the bacterium Corynebacterium glutamicum, 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.

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

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

(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.

(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).

In a preferred embodiment of the present invention, cells of Corynebacterium glutamicum, preferably C. glutamicum ATC13032, are used as microorganisms for providing 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 2023/111053.

(i) A reduced activity of the anthranilate phosphoribosyltransferase 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 natively present 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 which has a lower transcription activity than the endogenously present promoter sequence or by modifying the distance of the ribosome binding site to 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 the anthranilate phosphoribosyltransferase is reduced by deleting or inactivating the gene for the 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 defined in SEQ ID NO. 1 or 2, preferably SEQ ID NO. 2. The amino acid sequence of the anthranilate phosphoribosyltransferase preferably corresponds to the endogenous anthranilate phosphoribosyltransferase, particularly preferably it is defined by SEQ ID NO. 3 or a variant thereof.

(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. 6 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. 7 or a variant thereof. Increased expression of a gene can be achieved by all methods known to the person skilled in the art. This can be achieved, 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 enhanced expression of endogenous genes is Ptu/as defined in SEQ ID NO. 8.

(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. 9 or a variant thereof and SEQ ID NO. 10 or a variant thereof. This is preferably achieved by introducing additional copies of the genes encoding these enzymes into the microorganism. The Ptuf promoter is preferably used to control expression.

(iv) 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 derived from the product. Preferred is an enzyme having the amino acid sequence defined in SEQ ID NO. 11 or a variant thereof.

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.

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.

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

Preferred is the TrpG domain as defined by amino acid positions 3 to 196 of SEQ ID NO. 13 (TrpGD from E. coli) or a variant thereof. Also preferred are TrpG from Bacillus subtilis (SEQ NO. 14), the TrpG domain of TrpGD from Salmonella typhimurium (amino acid positions 3 to 196 of SEQ ID NO. 15), TrpG from Cupriavidus necator (SEQ ID NO. 16), TrpG from Corynebacterium glutamicum (SEQ ID NO. 17), or a variant of one of the aforementioned polypeptides.

Preferred is the TrpD domain as defined by amino acid positions 202 to 531 of SEQ ID NO. 13 (TrpGD from E. coli) or a variant thereof. Also preferred are TrpD from Bacillus subtilis (SEQ NO. 18), the TrpD domain of TrpGD from Salmonella typhimurium (amino acid positions 202 to 531 of SEQ ID NO. 15), TrpD from Cupriavidus necator (SEQ ID NO. 19), TrpD from Corynebacterium glutamicum (SEQ ID NO. 20) or a variant of one of the aforementioned polypeptides.

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. In this case, the same promoter can be used for the expression of both polypeptides.

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.

In the present application, a "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 position in the polypeptide. However, they preferably only occur at the N-terminus and/or C-terminus of the polypeptide. Amino acid substitutions are preferably conservative substitutions, i.e., substitutions in which the modified amino acid has a residue with similar chemical properties to the amino acid present in the unmodified 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.

Aminobenzoic acid occurs in three isomeric forms (ortho-, meta-, and para-aminobenzoic acid). In principle, the process according to the invention can be applied to all three isomers, either in isomerically pure form or as mixtures of different isomers. However, the preparation of ortho-aminobenzoic acid or para-aminobenzoic acid, in particular in isomerically pure form, is preferred. The preparation of ortho-aminobenzoic acid, in particular in isomerically pure form, is particularly preferred. In this context, "isomerically pure" means in the terminology of the present invention 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).

Regardless of which microorganism is used and which isomer is desired, the fermentation broth at the beginning of the fermentation in step (A) 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 aminobenzoic acid (present as an acid or its anion, depending on the pH value), the desired fermentation product.

Preferred fermentable carbon-containing compounds are those that can also serve as an energy and carbon source for the growth of the recombinant cells of the microorganism used. Particularly suitable for step (A) are starch hydrolysate, formic acid, alkali metal salts of formic acid, ammonium formate, acetic acid, alkali metal salts of acetic acid, ammonium acetate, sugar cane juice, sugar beet juice, hydrolysates from lignocellulose-containing raw materials, and mixtures of two or more of the aforementioned carbon-containing compounds. Acetic acid, formic acid, and their salts are preferably used not as the sole fermentable carbon-containing compound, but rather in a mixture with at least one of the other carbon-containing fermentable compounds.

Particularly suitable nitrogen-containing compounds are ammonia gas, ammonia water, ammonium salts, soy protein, urea and mixtures of two or more of the aforementioned nitrogen-containing compounds.

As already mentioned, the pH value to be maintained during fermentation depends on the microorganism used. Microorganisms such as Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum or Bacillus coagulans are preferably cultivated at "neutral to basic pH values" (i.e., in particular at a pH value in the range of 5.5 to 11, preferably 6.0 to 8.0). Preference is given to using Corynebacterium glutamicum, in particular Corynebacterium glutamicum ATCC 13032. When the fermentation is carried out at the stated pH values, the aminobenzoic acid produced is predominantly to completely in the form of its anion HzNCgH^OO'. In this case, the reduction product in step (C) is preferably selected from ammonium formate, an alkali metal salt of formic acid, ammonium acetate, or an alkali metal salt of acetic acid. As regards the fermentable carbon-containing compound, in this embodiment formic acid and acetic acid, if used at all, are preferably not used as such, but in the form of their salts. The fermentable carbon-containing compound, when fermented in the neutral to basic range, particularly includes starch hydrolysate, alkali metal salts of formic acid, ammonium formate, alkali metal salts of acetic acid, ammonium acetate, sugar cane juice, sugar beet juice, hydrolysates from lignocellulose-containing raw materials, and mixtures of two or more of the aforementioned carbon-containing compounds. Starch hydrolysate, sugar cane juice, sugar beet juice, and/or hydrolysates from lignocellulose-containing raw materials are preferred.

Microorganisms such as Ashbya gossypii, Pichia pastoris, Hansenula polymorpha, Kluyveromyces marxianus, Yarrowia lipolytica, Zygosaccharomyces bailii or Saccharomyces cerevisiae, on the other hand, are preferably cultivated in an acidic environment (i.e. in particular at a pH in the range of 3.0 to < 5.5, preferably 3.5 to 5.0). Depending on the specific pH value within the range of 3.0 to <5.5, the aminobenzoic acid is present in the fermentation broth as an anion (H2NC6H4COO) or in the electroneutral form (H2NC6H4COOH or H2TCeHziCOCr). (The formation of cations HsIXTCgH^OOH at pH values in the lowest part of the stated range, i.e., at pH 3.0 or slightly above, cannot be completely ruled out, but at most represents a negligible proportion of the total aminobenzoic acid present.) In this embodiment, the reduction product in step (C) is preferably selected from formic acid or acetic acid. As far as the fermentable carbon-containing compound is concerned, in this embodiment formic acid and acetic acid are used, if at all, preferably as such, and not preferably as salts. Thus, in fermentation in the acidic range, the fermentable carbon-containing compound includes, in particular, starch hydrolysate, formic acid, acetic acid, sugar cane juice, Sugar beet juice, hydrolysates from lignocellulose-containing raw materials, and mixtures of two or more of the aforementioned carbon-containing compounds. Starch hydrolysate, sugar cane juice, sugar beet juice, and/or hydrolysates from lignocellulose-containing raw materials are preferred.

In one embodiment of the invention, step (A) is carried out continuously, ie the reactants are continuously fed to the fermentation reactor and the product is continuously removed from the fermentation reactor. The product supplied to the fermentation reactor In the simplest case, the continuously withdrawn product is the fermentation broth containing aminobenzoate anions and/or aminobenzoic acid, including the microorganisms present therein. However, it is also conceivable to retain the microorganisms in the fermentation reactor using known separation processes (particularly filtration) and to withdraw a clarified fermentation broth containing aminobenzoate anions and/or aminobenzoic acid from it. Such clarification of the fermentation broth can, of course, also be carried out outside the fermentation reactor, in particular by filtration, centrifugation, or sedimentation.

In another embodiment of the invention, step (A) is carried out in a discontinuous process (so-called "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., aminobenzoic acid, and the complete or partial emptying of the reactor after completion of the fermentation. In a variant of the discontinuous mode (so-called "fed-batch mode"), the reactants are fed to the fermentation reactor (continuously or discontinuously [i.e., in portions]) for as long as the reactor volume allows, without any products being removed from the fermentation reactor—possibly with the exception of gaseous components that are discharged via a connection of the fermentation reactor to an exhaust system. 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 batch processes, clarification of the fermentation broth, particularly by filtration, centrifugation, or sedimentation, is preferred outside the fermentation reactor.

Separated microorganisms (biomass) can be returned to the fermentation process.

Regardless of the exact operating mode, the fermentation reactor preferably comprises 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 comprises devices for adjusting at least one (preferably all) of the aforementioned process parameters.

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). PROCESSING THE FERMENTATION BROTH

Step (B) of the process according to the invention relates to the recovery of aminobenzoic acid from the fermentation broth.

The execution of step (B) depends on the pH of the fermentation. If this is sufficiently above the isoelectric point of the aminobenzoic acid isomer to be obtained (pH 3.50 for ortho-aminobenzoic acid), the aminobenzoic acid is present predominantly or entirely as an anion and thus in dissolved form. In this case, it is preferable to first clarify the fermentation broth, i.e., to remove suspended solids, such as in particular the microorganisms used, whereby the aminobenzoate anions are present in dissolved form in the clarified fermentation broth thus obtained. Conventional methods such as filtration, centrifugation, or sedimentation are suitable for clarification. The aminobenzoic acid can then be obtained from the clarified fermentation broth using conventional methods, which are explained in more detail below.

If the pH of the fermentation is close to or significantly below the isoelectric point of the aminobenzoic acid isomer to be obtained, then at least a significant portion of the aminobenzoic acid is present as an acid (and not as its anion). Due to the low water solubility of aminobenzoic acid, it is then present, at least to a large extent, as a solid suspended in the fermentation broth. Here, too, the procedure can be such that the fermentation broth is first clarified as described above and the portions of the aminobenzoic acid or its anion dissolved in the clarified fermentation broth are isolated using conventional methods. However, since the majority of the aminobenzoic acid was obtained together with the microorganisms as a solid, the microorganisms must be separated from this solid. This can be achieved, for example, by treating (extracting) the solid with an aqueous base solution (e.g., sodium hydroxide or potassium hydroxide solution) or with a strong aqueous acid (hydrochloric acid or sulfuric acid), whereby the aminobenzoic acid is converted into its anion or cation and selectively dissolved. This is followed by solid-liquid separation using conventional methods, such as filtration, centrifugation, or sedimentation. The aminobenzoic acid can then be isolated from the resulting aqueous solution containing aminobenzoate anions or protonated aminobenzoic acid using conventional methods (crystallization by adjusting the pH to a value at or close to the isoelectric point). It is also conceivable to treat the fermentation broth directly, without prior clarification, with an aqueous base solution or aqueous acid.

Alternatively, the aminobenzoic acid present in the solid obtained during clarification of the fermentation broth can also be extracted with an organic solvent. It is also conceivable to extract the fermentation broth directly, without prior clarification, with an organic solvent. Suitable organic solvents include, for example, Cs-Cz-alkanols or aniline (the latter especially when Aminobenzoic acid is only an intermediate in aniline production. The aminobenzoic acid can be easily isolated by evaporating the resulting solution. Depending on the intended subsequent application, the resulting solution of aminobenzoic acid in the organic solvent can also be further processed directly (e.g., decarboxylated); such a procedure is covered by the formulation "Extraction of aminobenzoic acid from the fermentation broth."

In any case, it is preferable to proceed in such a way that the resulting solution (in the aqueous base or in the organic solvent) is as concentrated as possible.

The extraction of aminobenzoic acid from aqueous solutions of its anion (clarified fermentation broth or basic extract) is preferably carried out by crystallization. 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 this step. Separation can be carried out using conventional techniques, in particular by evaporation or membrane processes. All evaporation apparatus known in the art are suitable for evaporation. To minimize thermal stress, evaporation is preferably carried out 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 from 45 °C to 97 °C, in particular up to 82 °C.

A suitable technical apparatus, known in the art as a crystallizer, is used for crystallization. Suitable crystallizers include, for example, stirred tanks or forced-circulation crystallizers such as those of the "Oslo" type. In the crystallizer, the pH is adjusted to a value in the range of 3.0 to 4.7, preferably 3.2 to 3.7, particularly preferably 3.4 to 3.6, and most preferably 3.5. This is preferably done by adding an acid selected from hydrochloric acid, sulfuric acid, or phosphoric acid. This pH adjustment converts the aminobenzoic acid anions (H2NC6H4COO) predominantly to completely into the electroneutral form (H2NC6H4COOH or HsIXTCgH^OO) and causes them to crystallize out. This type of crystallization is also referred to as reactive crystallization. The crystallized aminobenzoic acid can be isolated by known methods, in particular filtration, sedimentation, or centrifugation, leaving behind an aqueous mother liquor.

It has proven effective to feed the fermentation broth and the acid to the crystallizer via spatially (as far as possible) separated feed devices. This ensures that the reactants are mixed as well as possible with the reactor contents before the acid-base reaction begins. Pipelines, preferably with shut-off valves, can be considered as feed devices. 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 wall. 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, wherein the feed devices are opposite one another and are located as close as possible, in particular directly, to the reactor wall.

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 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.

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).

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

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 at the selected temperature is significantly above the pH at which the minimum solubility of aminobenzoic acid is reached, the clarified fermentation product is initially only slightly acidified to a pH at which the minimum solubility of aminobenzoic acid is not yet reached at the selected temperature and under the given boundary conditions, but which is significantly closer to this pH (preferably, acidification to pH 5.0 to 6.5 is carried out in this first step). 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 a comparatively low solubility and therefore promotes the most complete recovery of the aminobenzoic acid possible. The amount of seed crystals added is preferably about 0.1% to 1% of the aminobenzoic acid dissolved in the fermentation broth. In this way, a suspension of seed crystals is obtained (see also WO 2017/085170 A1). The pH is then adjusted to 3.0 to 4.7, preferably 3.2 to 3.7, particularly preferably 3.4 to 3.6, and most preferably 3.5, by adding acid (in the case of prior slight acidification, the same acid as used there). The acid is preferably added slowly; for example, at 1 kg initially introduced fermentation broth and using 37% hydrochloric acid within 1 h. After the acid addition is complete, stirring is continued for a certain period of time, in particular for the same period of time that the addition of the acid after addition of the seed crystals took. The precipitated aminobenzoic acid is then separated by filtration (if necessary under vacuum), sedimentation or centrifugation (preferably by centrifugation) and preferably washed several times (in particular twice) with an aqueous acidic washing liquid (in particular the same acid that was used for precipitation) with a pH of 3.0 to 4.7, preferably 3.2 to 3.7, particularly preferably 3.4 to 3.6, very particularly preferably 3.5. Depending on the purity requirements of the intended subsequent application, the aminobenzoic acid can also be purified by recrystallization.

In continuous crystallization, seed crystals generally only need to be added specifically during the start-up of the continuous process, as additional seed crystals later form spontaneously in situ (so-called secondary nucleation). The seed crystal suspension 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.

In addition to clarification, the fermentation broth can be subjected to further pretreatment steps before being subjected to crystallization. Of particular note here is decolorization of the (especially already clarified) fermentation broth. Such decolorization is preferably carried out by passing the (preferably clarified) fermentation broth 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).

CONVERSION OF CARBON DIOXIDE TO A REDUCTION PRODUCT

Step (C) of the process according to the invention comprises the conversion of carbon dioxide to a reduction product selected from formic acid, ammonium formate, an alkali metal salt of formic acid, acetic acid, ammonium acetate or an alkali metal salt of acetic acid.

In principle, the carbon dioxide used in step (C) can originate from any source, provided that it is obtained there in a purity required for the reduction or can be brought to such a purity with reasonable effort.

Process-immanent sources for the carbon dioxide are preferred, whereby a process-immanent source in this context means in particular the further processing of the aminobenzoic acid to a direct reaction product thereof with the formation of carbon dioxide. Although the use of carbon dioxide formed in the fermentation according to step (A) is technically possible, it is not preferred because this carbon dioxide is obtained in high dilution and the effort required to concentrate it would be disproportionately high.

Carbon dioxide resulting from the further processing of aminobenzoic acid into a direct reaction product is, in particular, the carbon dioxide produced during the decarboxylation of aminobenzoic acid to aniline (see below for further details). This carbon dioxide from the decarboxylation is particularly suitable because it contains at most trace amounts of aniline or anthranilic acid as impurities, which can be easily separated.

As already mentioned, the carbon dioxide used for the reduction can also come from an external source. The use of process-immanent and external sources can also be combined, i.e., the carbon dioxide used can be a mixture of carbon dioxide from a process-immanent source, such as decarboxylation, and carbon dioxide from an external source. Various sources are conceivable as external sources, such as carbon dioxide from

• a process for the production of (i) bioethanol, (ii) cement, (iii) hydrogen (e.g. from steam reforming including CO conversion or microbial electrolysis [3]) or (iv) epoxides, or

• a biological degradation process carried out in a digester (in particular in a sewage treatment plant or biogas plant), or

• from combustion of fuels, or

• a process for extracting carbon dioxide from air (so-called direct air capture).

(For [3] see: Abudukeremu Kadier et al., "A comprehensive review of microbial electrolysis cells (MEC) reactor designs and configurations for sustainable hydrogen gas production", published in Alexandria Engineering Journal 2016, 55, 427 - 443.)

In a preferred embodiment of the process according to the invention, such carbon dioxide is extracted from an external carbon dioxide source using known adsorption, absorption, and/or membrane processes. Such processes are also suitable for extracting carbon dioxide from the exhaust gas of the fermentation process.

The separation of carbon dioxide from gas mixtures is achieved, for example, by pressure swing adsorption (PSA) on activated carbon, molecular sieves, and carbon molecular sieves. This is based on the different adsorption behavior of the gas components towards the adsorbent (the solid and stationary phase on which the adsorptive, i.e. the gas components, are bound). A Amine scrubbing. This is an absorption process in which the chemical reaction between gas components and the solution overrides the physical absorption, allowing even more carbon dioxide to be absorbed. An amine solution serves as the scrubbing agent. Membrane processes use diffusion membranes, which exploit the different solubility of the gas components in the membrane.

Carbon dioxide separation from ambient air (so-called direct air capture, DAC) can also be used to provide carbon dioxide for further processing in the process according to the invention. The most important DAC approaches pursued to date can be fundamentally divided into the three technology paths outlined below:

Absorption and electrodialysis is a process in which the carbon dioxide contained in the intake air is absorbed using sodium hydroxide solution. By acidifying the resulting sodium carbonate solution with sulfuric acid, the carbon dioxide is removed in a nearly pure form. The sodium hydroxide solution and sulfuric acid are then regenerated using an electrochemically driven membrane process (electrodialysis).

In the absorption and calcination process, carbon dioxide is absorbed similarly to the previous process using an alkali metal hydroxide solution (sodium hydroxide or potassium hydroxide). When potassium hydroxide is used, the aqueous potassium (hydrogen) carbonate resulting from carbon dioxide absorption is precipitated into calcium carbonate in a pellet reactor and decomposed into carbon dioxide and calcium oxide by calcination. The latter is hydrated to calcium hydroxide and is then available for recycle.

In the adsorption and desorption process, the carbon dioxide is first bound to a sorbent via organic chemisorption, which is then regenerated primarily by the addition of heat or moisture. The filter material used can include dry cellulose, on whose surface amine compounds are deposited, or a resin with amines deposited on it.

The actual conversion of carbon dioxide to the reduction product is preferably carried out by reaction with hydrogen or electrolytically by means of processes known per se and therefore only briefly described below.

The reduction of carbon dioxide with hydrogen to formic acid or its salts is carried out 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") [4]. See, for example, Scheme 2 (formic acid) and Scheme 3 (formate salt) of [4]. A process for the production of formic acid by CO2 hydrogenation using ruthenium catalysts, in particular [Ru(H)2(PnBus)4], using amines, in particular trihexylamine, and polar (hydrogen-bonding) solvents such as diols (in particular 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") [5]. 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.

The reaction of carbon dioxide with hydrogen to form formic acid can also be achieved biotechnologically; see, for example, F. Oswald et al., ("Formic acid formation by Clostridium ljungdahlii at elevated pressures of carbon dioxide and hydrogen"), Front. Bioeng. Biotechnol. 2018, Volume 6, Article 6 [6],

The electrochemical reduction of carbon dioxide to formic acid 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, C. M.S. Lobo, S. Baranyai, T. Khoza, M. Auinger, and E. Klemm in Journal of CO Utilization 2022, 56, 101823 ("Producing formic acid at low pH values by electrochemical CO2 reduction") [7].

The electrochemical reduction of carbon dioxide to formate salts occurs at pH values above the pKa value of formic acid, for example at pH 10. Suitable electrolysis cells and operating modes for this purpose are described by Achim Löwe et al. in ACS Sustainable Chem. Eng. 2021, 9, 4213 - 4223 ("Optimizing Reaction Conditions and Gas Diffusion Electrodes Applied in the CO2 Reduction Reaction to Formate to Reach Current Densities up to 1.8 A cm~ ² ") [8].

The electrochemical reduction of carbon dioxide to acetic acid is described by Ratnadip De et al. in Angew. Chem. Int. Ed. 2020, 59, 10527–10534 ("Electrocatalytic Reduction of CO2 to Acetic Acid by a Molecular Manganese Corrole Complex") [9]. Another conceivable route is the reduction of CO2 (particularly by catalytic hydrogenation) to methanol and its use in a Monsanto process for the production of acetic acid by the carbonylation of methanol with carbon monoxide. The carbon monoxide required for the carbonylation is also accessible by catalytic hydrogenation or electrochemical reduction (see, for example, WO 2021/069498 A1).

3990 [10]. Acetate can be obtained by reacting CO2 or mixtures of CO2 and CO with hydrogen in a biotechnological process; see the already mentioned publication Microbial Biotechnology 2018, 11 (4), 606 - 625 [1], e.g. Fig. 2 therein. There are also electrochemical processes, see for example HH Heenen et al., "The mechanism for acetate formation in electrochem with potential, pH, and nanostructuring", published in

3990 [10].

ADDITION OF THE REDUCTION PRODUCT TO THE FERMENTATION

Step (D) of the process according to the invention involves adding the reduction product from step (C) to the fermentation of step (A). The recycled reduction product serves as a carbon source for the microorganisms in the fermentation and thus reduces the need for primary fermentation substrate, e.g., sugar (see also the exemplary embodiments for the use of acetate). If the fermentation takes place at pH values at which the aminobenzoic acid is obtained predominantly or entirely as the aminobenzoate anion (e.g., as sodium aminobenzoate), and if a (particularly alkali metal) formate or acetate is used as the reduction product, the additional advantage arises that the counterions of the formate or acetate salts can serve as counterions of the fermentation product, the aminobenzoate anion. In this way, the pH is stabilized, and the need for additional base (such as sodium hydroxide solution) for pH adjustment is reduced or completely avoided.

This ultimately leads to the following advantages: (1) Lower manufacturing costs due to lower substrate and base consumption in the production of the target product; (2) lower CO2 footprint of the target product aminobenzoic acid, as fewer raw materials such as sugar and base are required (furthermore, in the case of further processing to aniline, there is the possibility of recycling the CO2 produced during decarboxylation, possibly supplemented with CO2 from other sources, and thus not emitting it).

To illustrate the advantage of substrate savings, the influence of the addition of the reduction product on the theoretical yield was calculated. The theoretical yield is expressed as the maximum achievable mass of anthranilic acid [g] per mass of glucose [g] used, or per mass of organic acid [g] used if no glucose is used. Elemental balances (balances for carbon atoms, nitrogen atoms, and hydrogen atoms) and charge balances were established to calculate this yield (see, for example, Gregory N. Stephanopoulos et al., "Metabolic Engineering, Principles and Methodologies," 1998, Elsevier Inc., https://doi.org/10.1016/B978-0-12-666260-3.X5000-6, Chapter 10.1.2 [11]). The reactants were glucose and the respective organic acid, either alone or in specified molar ratios, as well as ammonia and molecular oxygen. Anthranilic acid, water, and carbon dioxide were permitted as products, with the amount of anthranilic acid maximized and the formation of water and carbon dioxide serving to balance excess hydrogen and oxygen. For example, using glucose and formic acid in a molar ratio of 1:1 (see also entry 4 of Table 1 below), the reaction equation is: 14 C ₆ HI ₂ O ₆ + 14 CHOOH + 13 NH ₃ -> 13 H ₂ N(C ₆ H ₄ )COOH + 72 H ₂ O + 7 CO ₂ .

Metabolic pathways and thermodynamics were not considered in the calculation. The results are summarized in Table 1.

Table 1: Effect on the yield of anthranilic acid (oAB) by the process according to the invention

Explanations:

SdT = prior art procedure; Ref. = reference; erf.-gem. = inventive procedure. As can be seen from Table 1, the amount of glucose can be reduced using the inventive procedure without having to expect a deterioration in yield.

The reduction product can be added to the fermentation continuously or intermittently. It is important to ensure that the reduction product concentration in the fermentation broth (which, as mentioned above, serves as an additional carbon source for the microorganisms) remains below a concentration that is toxic to the microorganisms. The limit value varies depending on the microorganism and the type of reduction product added and can be easily determined in preliminary tests.

For the utilization of formate by microorganisms, see also Justine Turlin et al., Metabolie Engineering 2022, 74, 191 - 205, "Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation" [12] Formate assimilation is further described in WO 2021/116330 Al.

USE OF AMINOBENZOIC ACID IN THE PRODUCTION OF OTHER VALUE-PRODUCED PRODUCTS

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

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 of indigo, as well as for pharmaceuticals and pesticides (acaricides). Anthranilic acid produced according to the invention is suitable for all of these purposes.

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 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.

Aminobenzoic acid obtained according to the invention can also be decarboxylated to aniline, which is an important raw material, particularly in the polyurethane industry.

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. DECARBOXYLATION OF AMINOBENZOIC ACID

As already mentioned, in a particularly preferred embodiment, the process according to the invention comprises the decarboxylation of the aminobenzoic acid to aniline.

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

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-AlO 2 or p-Al 2 O 3 , 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.

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.

The decarboxylation can also take place 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, catalysts external to the system 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, from 0.1% to 90%, preferably 1.0% to 70%, particularly preferably 5.0% to 50%.

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.

As far as the reaction procedure is concerned, both gas phase and liquid phase are suitable. The reaction can be carried out continuously (preferred) or discontinuously.

Preferred procedures include carrying out the decarboxylation of the aminobenzoic acid

• 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),

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

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

A tubular reactor is defined as a tubular reactor through which the reacting reaction mixture flows during operation in continuous reaction mode (which is preferred). Tubular reactors with small length-to-diameter ratios are also referred to as tower reactors; these are also included in the definition of the term "tubular reactor" used here.

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

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.

The aniline formed can be isolated and purified by standard techniques, particularly distillation, and used as described above. Examples:

Influence of the addition of potassium acetate on the yield of anthranilic acid

Tribe description

All experiments were performed with the strain described below.

Based on the bacterium Corynebacterium glutamicum ATCC13032, a microbial strain producing anthranilic acid was generated through targeted chromosomal modifications. All genetic modifications, i.e., chromosomal deletions and gene integration, were performed by double homologous recombination using corresponding pK19mobsacß derivatives (Schäfer et al., 1994: "Small mobilizable multipurpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum."; Gene 1994, 145 (1), 69–73 [13]; doi: 10.1016/0378-1119(94)90324-7)

The activity of the anthranilate phosphoribosyltransferase TrpD was reduced by first deleting the native trpD-A\\e\ 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. 1).

The gene (SEQ ID NO. 5) encoding one or the only phosphoenolpyruvate carboxylase in C. glutamicum (SEQ ID NO. 4) was deleted.

To enhance the aromatic biosynthesis pathway, an artificial, polycistronic P _tU f- aroLAC operon (SEQ ID NO. 12), consisting of the genes aroL (b0388) from Escherichia coli, as well as aroA (cg0873) and aroC (cgl829) from C. glutamicum, was integrated downstream of cg2563 under the control of the constitutive promoter of the elongation factor Tuf. In addition, the allele aroG ^new , which encodes a feedback-resistant variant of the DAHP synthase (SEQ ID NO. 11) from E. coli, was integrated downstream of cg3132 into the genome of the strain.

Procedure

Comparative cultivations of the above-mentioned strain were carried out for the production of ortho-aminobenzoic acid starting from 20 g/L D-glucose and different concentrations of potassium acetate. Table 2: Overview of the concentrations of the carbon sources D-glucose and potassium acetate in the main culture media. Two replicates were prepared for media 1 to 4, each with an initial culture volume of 50 mL.

Two replicates were created for each condition in Table 2. The cultivation conditions are shown below (Table 3).

Table 3: Overview of the cultivation conditions for the pre- and main cultures. The second pre-cultures, as well as the main cultures (initially 50 mL each before the first sample collection), were carried out in Erlenmeyer flasks with a maximum capacity of 1000 mL each. Cotton plugs served as a sterile barrier.

Media used, their composition and production

Unless otherwise stated, all media are prepared with ultrapure water (H2O MilliQ) and autoclaved.

Tabelle 5: Flüssiges Minimalmedium mit Komplexbestandteilen für die Anzucht von Zellen in der Vorkultur. Die Mengen der Einwaage sind für 1 L fertiges CGXII Vorkulturmedium angegeben. Die final angestrebten Konzentrationen im fertigen Medium werden erst nach vollständiger Supplementation erreicht.

Table 4: Liquid and solid complex media from brain-heart infusion (BHI) for cell culture.

Table 5: Liquid minimal medium with complex components for cell growth in preculture. The sample weights are given for 1 L of finished CGXII preculture medium. The final target concentrations in the finished medium are only achieved after complete supplementation.

MOPS = (3-(N-morpholino)propanesulfonic acid)

Table 6: Liquid minimal medium for the main cultures. The sample weights are given for 1 L of finished CGXII main culture medium. The final target concentrations in the finished medium are only achieved after complete supplementation.

Ta bei le 8: Übersicht zur Herstellung der 600 g/L d-Glucose-Stammlösung.

Table 7: Overview of the preparation of the 2 g/L biotin stock solution. The solution is not autoclaved, but sterile filtered (0.2 μm). The solution can be stored for 1 month at 4 °C.

Table 8: Overview of the preparation of the 600 g/L d-glucose stock solution.

Tabelle 10: Übersicht zur Herstellung der 10 g/L CaCh-Stammlösung.

Table 9: Overview of the preparation of the 327 g/L potassium acetate stock solution.

Table 10: Overview of the preparation of the 10 g/L CaCh stock solution.

Table 11: Overview of the preparation of the 200 g/L MgSO ₄ stock solution.

Tabelle 13: Übersicht zur Herstellung von lx Phosphatpuffer (PBS). Verwendet wurde der lOx PBS (Artikelnummer BP399-1) der Firma Fisher Scientific GmbH.

Table 12: Overview of the preparation of the trace element stock solution. The solution is not autoclaved, but sterile filtered (0.2 μm). This solution is stable for 6 months at 4 °C. Due to the small sample weights, we recommend preparing a 1 L batch.

Table 13: Overview of the preparation of lx phosphate buffer (PBS). The lOx PBS (article number BP399-1) from Fisher Scientific GmbH was used.

Devices used

Tabelle 14 (fortgesetzt)

Table 14: Overview of the parameters investigated in this study and the equipment and procedures used to investigate them.

Table 14 (continued)

Implementation

To generate starter cultures, cell mass was taken from glycerol-surviving forms of the microbial cultures used for cultivation. This was used to inoculate a BHI agar plate. The BHI agar plates were incubated at 30 °C above atmospheric humidity.

The agar plate culture prepared in this way was used to inoculate a BH I liquid culture. For this purpose, a small amount of cell mass was taken from the respective BHI agar plate cultures and inoculated with 4.5 mL of BHI liquid medium in a round-bottom tube. The liquid cultures were incubated for 7.5 h at 30 °C and 200 rpm (preculture I).

For the second preculture, CGXII preculture medium containing 20 g/L D-glucose was used. Two shake flasks containing 49 mL of the preculture medium were transferred to a 1000 mL Erlenmeyer flask, 1 mL of the BHI liquid preculture was added, and these shake flask precultures were incubated at 200 rpm and 30 °C for 17 h.

Preculture II was used for inoculating the main cultures. After determining the optical density, both cultures were first centrifuged completely, and the resulting cell pellets were resuspended in PBS buffer. An optical density of 50 was set. The suspensions were combined, and all main cultures were inoculated with 1 mL of the same cell suspension to an initial optical density (OD 500 ) of 1. Two cultures were established for each main culture condition. Table 15: Measurement of the optical density (ODgoo) after incubation of the second preculture in the shake flask and volumes required for resuspension of the cell pellets.

The main cultures prepared in this way were transferred to a Kühner incubation shaker (Table 14) and incubated. For sampling throughout the cultivation period, the shake flasks were removed from the incubation shaker. Samples were taken under sterile conditions for the determination of glucose, acetate, ammonium, ortho-aminobenzoic acid, ODgOO, and dry biomass. Results

Acetate concentration

Table 16: Overview of the measured acetate concentrations over time for two replicates each with medium 1 to 3. The samples with medium 3 were not measured because no acetate was added.

(nb = not determined; DL = detection limit) Ammonium concentration

D-Glucose-Konzentration Table 17: Overview of the ammonium concentrations (NH concentration) over time for two replicates each with medium 1 to 3.

D-glucose concentration

Biotrockenmasse-Konzentration (BTM) Table 18: Overview of the measured d-glucose concentrations over time for two replicates each with medium 1-3. The detection limit (DL) was 0.3 mmol/L D-glucose.

Biomass dry matter concentration (BDM)

ortho-Aminobenzoesäure-Konzentration (oAB) Tabelle 20: Übersicht über die ortho-Aminobenzoesäure-Konzentration über den zeitlichen Verlauf für je zwei Replikate mit Medium 1 - 3. Das Detektionslimit (DL) betrug 0,2 g/L

Table 19: Final dry biomass concentrations for two replicates each with medium 1 to 3.

ortho-Aminobenzoic acid concentration (oAB) Table 20: Overview of the ortho-Aminobenzoic acid concentration over time for two replicates each with medium 1 - 3. The detection limit (DL) was 0.2 g/L

Product yield

Table 21: Overview of the calculated ortho-aminobenzoic acid yields after 30 h and 52 h for two replicates with medium 1 to 3, based on glucose. These are expressed as mass-related (gAb/gC-glucose) and molar-related quotients (mol _Ab /mol _Glucose ).

Summary

In the shake flask cultivation of an ortho-aminobenzoic acid producer based on C. glutamicum described here, the influence of different concentrations of (potassium) acetate as an additional C source on the yield of ortho-aminobenzoic acid based on the amount of glucose used was investigated.

It was found that the final oAB titer and thus the oAB yield relative to the amount of glucose used (g/L glucose) increased with increasing acetate concentration. Of the media tested, the highest yield was achieved with Medium 2, which contained 20 g/L glucose and 13.299 g/L potassium acetate. A value of 0.186 g/L glucose was achieved, whereas without acetate (Medium 3) a value of only 0.109 g/L glucose was achieved.

Claims

Patent claims: 1. A process comprising the preparation of aminobenzoic acid, the process comprising the following steps: (A) fermenting a fermentable carbon-containing compound and a nitrogen-containing compound in the presence of microorganisms to obtain a fermentation broth containing aminobenzoate anions and/or aminobenzoic acid; (B) recovering aminobenzoic acid from the fermentation broth; (C) converting carbon dioxide to a reduction product selected from formic acid, ammonium formate, an alkali metal salt of formic acid, acetic acid, ammonium acetate or an alkali metal salt of acetic acid; and (D) Addition of the reduction product from step (C) to the fermentation of step (A). 2. The process according to claim 1, wherein carbon dioxide formed in the fermentation of step (A) is not converted into the reduction product in step (C). 3. A process according to claim 1 or 2, wherein ortho-aminobenzoic acid or para-aminobenzoic acid is produced. 4. A process according to any one of claims 1 to 3, wherein the process comprises a step (E) in which the aminobenzoic acid from step (B) is decarboxylated to aniline and carbon dioxide formed thereby is converted in step (C) to the reduction product. 5. A process according to any one of claims 1 to 4, wherein in step (C) carbon dioxide from an external source is converted to the reduction product. 6. A method according to claim 5, wherein the carbon dioxide is supplied from an external source of • a process for producing (i) bioethanol, (ii) cement, (iii) hydrogen or (iv) epoxides, or • a biological degradation process in a digester, or • combustion of fuels, or • a process for extracting carbon dioxide from air. 7. A process according to any one of claims 1 to 6, wherein step (C) comprises reacting the carbon dioxide with hydrogen or electrolyzing the carbon dioxide. 8. The method according to any one of claims 1 to 7, wherein the microorganisms in step (A) are selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans, Ashbya gossypii, Pichia pastoris, Hansenula polymorpha, Kluyveromyces marxianus, Yarrowia lipolytica, Zygosaccharomyces bailii, Saccharomyces cerevisiae or a mixture of two or more of the aforementioned microorganisms, and/or wherein the fermentable carbon-containing compound is selected from starch hydrolysate, formic acid, an alkali metal salt of formic acid, ammonium formate, acetic acid, an alkali metal salt of acetic acid, ammonium acetate, 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, and/or 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. 9. The method according to any one of claims 1 to 8, wherein the microorganisms in step (A) are selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans or a mixture of two or more of the aforementioned microorganisms. 10. The process according to claim 9, wherein the reduction product in step (C) is selected from ammonium formate, an alkali metal salt of formic acid, ammonium acetate or an alkali metal salt of acetic acid. 11. A process according to claim 9 or 10, wherein step (A) is carried out in a pH range of 5.5 to 11 to obtain a fermentation broth containing aminobenzoate anions. 12. A process according to any one of claims 9 to 11, wherein the fermentable carbon-containing compound is selected from starch hydrolysate, an alkali metal salt of formic acid, ammonium formate, an alkali metal salt of acetic acid, ammonium acetate, sugar cane juice, sugar beet juice, hydrolysates of lignocellulosic raw materials or a mixture of two or more of the aforementioned carbon-containing compounds. 13. The method according to any one of claims 1 to 8, wherein the microorganisms in step (A) are selected from Ashbya gossypii, Pichia pastoris, Hansenula polymorpha, Kluyveromyces marxianus, Yarrowia lipolytica, Zygosaccharomyces bailii, Saccharomyces cerevisiae or a mixture of two or more of the aforementioned microorganisms. 14. The process according to claim 13, wherein the reduction product is selected from formic acid or acetic acid. 15. A process according to claim 13 or 14, wherein step (A) is carried out in a pH- Range from 3.0 to < 5.5 to obtain an aminobenzoic acid and/or fermentation broth containing aminobenzoate anions. 16. The method according to any one of claims 13 to 15, wherein the fermentable carbon-containing compound is selected from starch hydrolysate, formic acid, acetic acid, 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.