Production of aminobenzoic acids

Abstract

The present invention relates to a process comprising aminobenzoic acid production, said process having the steps of: (A) fermenting a fermentable carbon- and 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

[0001] The present invention relates to a method for producing aminobenzoic acid, the method comprising: (A) fermenting a fermentable carbonaceous compound and a nitrogenous compound in the presence of microorganisms to obtain a fermentation broth containing an aminobenzoate anion and / or aminobenzoic acid; (B) obtaining 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 of step (A).

[0002] The production of organic acids via fermentation has recently received particular attention. Among the organic acids obtainable through fermentation, aminobenzoic acid, as an economically important product, deserves special emphasis. Aminobenzoic acid can be used, for example, in the production of dyes, fragrances, crop protectants, or pharmaceuticals. Another example of the use of aminobenzoic acid is its application in the production of aniline via decarboxylation. Aniline is particularly important as an intermediate in the production of isocyanates. The ortho-isomer of aminobenzoic acid, anthranilic acid, can also be used as a starting material for the production of poly(o-aminobenzoamide), as well as polyamines and the corresponding polyisocyanates. For example, according to WO2022 / 008450A1, anthranilic acid can be converted to anthranilic esters, which can then be catalytically converted to produce poly(o-aminobenzoamide) by eliminating the parent alcohol of the anthranilic ester through polycondensation.

[0003] The fermentation production of aminobenzoic acid is generally known in the prior art; see, for example, International Patent Application WO2015 / 124687 A1 (which describes a two-step production of aniline using an-aminobenzoic acid as an intermediate) and the references cited therein. The fermentation process is carried out in an aqueous medium, and in the case of producing aminobenzoic acid, it typically produces an aqueous product mixture with an aminobenzoic acid content particularly in the range of 10.0 g / L to 100 g / L. Fermentation liquid ).

[0004] Of particular importance is the ortho-isomer of aminobenzoic acid (annaminobenzoic acid). In bacterial and yeast metabolism, annaminobenzoic acid is formed in the shikimic acid pathway as a natural intermediate in tryptophan synthesis. In the biotechnological production of annaminobenzoic acid, its conversion in metabolic pathways is reduced or inhibited to achieve accumulation in fermentation media. This concept for the bioproduction of annaminobenzoic acid and its subsequent catalytic conversion to aniline is described in international patent application WO2015 / 124686A1 and the previously mentioned WO2015 / 124687A1. The described possible recombinant microorganisms are bacteria from the families Corynebacteria or Pseudomonad. A more recent application (WO2017 / 102853A1) describes the use of yeast.

[0005] Para-aminobenzoic acid has also attracted attention. Para-aminobenzoic acid can be synthesized in bacteria and yeast via the intermediate chorismate, which is formed as an intermediate in the shikimic acid pathway. The chorismate is first enzymatically converted to 4-amino-4-deoxychorismate, and then converted to para-aminobenzoic acid via another enzymatic reaction. A concept for the biotechnological production of aniline via the intermediate para-aminobenzoic acid is described in international application US2016 / 068876A1. Also described herein is a possible recombinant microorganism, among other things, using bacteria from the Corynebacterium family as a possible recombinant microorganism.

[0006] Fermentation processes typically produce waste gas containing carbon dioxide. This applies to both aerobic and anaerobic processes (such as bioethanol production). In most cases, existing technologies do not involve the further utilization of this carbon dioxide. EP3715464B1 describes a method in which microorganisms are cultured in a bioreactor, CO2 is collected from the bioreactor and reduced to organic starting materials, and at least a portion of the organic starting materials are fed into the bioreactor.

[0007] Similarly, carbon dioxide can be produced in downstream reactions of fermentation, such as the production of aniline from aminobenzoic acid mentioned above.

[0008] Ralf Takors and others Microbial Biotechnology 2018, 11 (4), 606 – 625 (“ Using gas mixtures of CO, CO 2 and H 2 as microbial substrates: the do's and don'ts of successful technology transfer from laboratory to production scale The use of a gaseous mixture of CO, CO2, and H2 for gaseous fermentation is described in [1] (see Figure 2 from [1]). Markus Stöckl et al. in ChemSusChem 2020, 13 , 4086 – 4093 ( From CO 2 to Bioplastic - Coupling the Electrochemical CO 2 Reduction with a Microbial Product Generation by Drop-in Electrolysis” [2] describes the electrochemical reduction of CO2 to formate and its application to the microbial insecticide copper-loving bacteria ( Cupriavidus necator Formate was used as the sole substrate for the production of polyhydroxybutyrate.

[0009] The biotechnology production of aminobenzoic acid can make a significant contribution to resource conservation and emission reduction. Previously, there were no documented methods that could reuse carbon dioxide formed during fermentation or otherwise produced within the fermentation process itself, thereby making it more sustainable.

[0010] Therefore, further improvements are needed in the fermentation production of aminobenzoic acid. Specifically, it is hoped that (i) can be utilized. Formed during fermentation (ii) Formed in any subsequent steps and / or (iii) From external sources carbon dioxide.

[0011] In view of this need, the present invention provides a method for producing aminobenzoic acid, the method comprising the following steps: (A) Fermenting (preferably aerobic) fermentable carbon and nitrogen compounds in the presence of microorganisms to obtain a fermentation broth containing aminobenzoate anions and / or aminobenzoic acid; (B) Obtaining aminobenzoic acid from the fermentation broth; (C) Convert carbon dioxide into a mixture selected from formic acid and ammonium formate (ammonium salt of formic acid [NH4+]). + [salts]), alkali metal salts of formic acid (especially sodium or potassium salts), acetic acid, ammonium acetate (ammonium salts of acetic acid [NH4] + The reduction product of alkali metal salts (especially sodium or potassium salts) of acetic acid or acetic acid, preferably acetic acid, ammonium acetate or alkali metal salts of acetic acid, particularly ammonium acetate or alkali metal salts of acetic acid; as well as (D) Add the reduction product from step (C) to the fermentation in step (A).

[0012] Completely unexpectedly, it has been found that formic acid, acetic acid, or their salts (preferably acetic acid and its salts, especially salts of acetic acid) produced by the reduction of carbon dioxide are suitable for feeding and use in fermentation for the production of aminobenzoic acid.

[0013] All in the context of this invention pH valueAll of these are related to the temperature at which the corresponding steps (e.g., step (A)) are performed, and can be easily measured using a glass electrode.

[0014] In the context of this invention, fermentable carbonaceous compounds should be understood to refer to any organic compound or mixture of organic compounds that can be used to produce aminobenzoic acid by means of the microorganisms used. The production of aminobenzoic acid can occur under aerobic (aerobic) or anaerobic (anaerobic) conditions, preferably under aerobic (aerobic) conditions, especially in the form of an oxygen-containing gas such as air.

[0015] The reduction products of carbon dioxide should be understood as those products of carbon dioxide reduction in which the oxidation number of the carbon atom corresponding to the carbon atom in carbon dioxide has been reduced compared to that of carbon dioxide. According to the present invention, the reduction products are selected from formic acid, ammonium formate, alkali metal salts of formic acid, acetic acid, ammonium acetate, or alkali metal salts of acetic acid.

[0016] Carbon dioxide in the context of this invention external sources It should be understood as referring to different The source of aminobenzoic acid produced through fermentation or as a direct reaction product. Aminobenzoic acid... direct reaction products This refers to a product that can be obtained directly (i.e., without intermediates) by reacting aminobenzoic acid. In this sense, direct reaction products It is aniline formed by decarboxylation.

[0017] First, various possible embodiments of the present invention will be discussed. Brief Overview as follows: In a first embodiment of the invention, which can be combined with all other embodiments, the carbon dioxide formed during fermentation in step (A) is not converted into the reduction product in step (C).

[0018] In a second embodiment of the invention, which can be combined with all other embodiments (except those limited to forming m-aminobenzoic acid or para-aminobenzoic acid), the method of the invention produces o-aminobenzoic acid.

[0019] In a third embodiment of the invention, which can be combined with all other embodiments (except those limited to forming m-aminobenzoic acid or o-aminobenzoic acid), p-aminobenzoic acid is produced in the method of the invention.

[0020] In a fourth embodiment of the present invention, which is a second specific embodiment, anthranilic acid is converted into poly(anthranilamide).

[0021] In a fifth embodiment of the invention, which is a second specific embodiment, anthranilic acid is converted into anthranilic acid derivative selected from anthranilic halide, isatoic anhydride, or mixtures thereof, and the anthranilic acid derivative is reacted with a polyol to form a polyamine.

[0022] In a sixth embodiment of the invention, which is a specific embodiment of the fifth embodiment, phosgenated polyamine is used to form polyisocyanate.

[0023] In the seventh embodiment of the invention, which can be combined with all other embodiments (except those excluding those in which aniline is formed from aminobenzoic acid), and particularly advantageously combined with the second embodiment, the method includes step (E), in which aminobenzoic acid from step (B) is decarboxylated to aniline and the resulting carbon dioxide is converted into the reduction product in step (C).

[0024] In the eighth embodiment of the invention, which is the seventh embodiment, aniline is reacted with formaldehyde to form methylene diphenylene diamine and polymethylene polyphenylene polyamine.

[0025] In a ninth embodiment of the invention, which is the eighth specific embodiment, phosgenation of methylene diphenylene diamine and / or polymethylene polyphenylene polyamine is used to form methylene diphenylene diisocyanate and / or polymethylene polyphenylene polyisocyanate.

[0026] In the tenth embodiment of the present invention, which is the ninth embodiment, methylene diphenyl diisocyanate and / or polymethylene polyphenyl polyisocyanate are reacted with a polyol to form a polyurethane.

[0027] In the eleventh embodiment, which is another specific embodiment of the invention as the seventh embodiment, aniline is converted into an azo compound.

[0028] In a twelfth embodiment of the invention, which can be combined with all other embodiments, carbon dioxide from an external source is converted into the reduction product in step (C).

[0029] In the thirteenth embodiment of the present invention, which is the twelfth embodiment, the carbon dioxide originates from the following external source: Processes used for the production of (i) bioethanol, (ii) cement, (iii) hydrogen (particularly for ammonia synthesis), or (iv) epoxides, or Biodegradation processes in digesters (especially in wastewater treatment plants or biogas plants), or From fuel combustion, or The process used to extract carbon dioxide from the air (so-called Direct air capture ).

[0030] In a fourteenth embodiment of the invention, which can be combined with all other embodiments, step (C) includes the reaction of carbon dioxide with hydrogen or the electrolysis of carbon dioxide.

[0031] In the fifteenth embodiment of the invention, which can be combined with all other embodiments (except those requiring a narrower selection of carbon-containing compounds), the fermentable carbon-containing compound is selected from starch hydrolysate, formic acid, alkali metal salts of formic acid, ammonium formate, acetic acid, alkali metal salts of acetic acid, ammonium acetate, sugarcane juice, beet juice, hydrolysate of lignocellulose raw material, or a mixture of two or more of the above-mentioned carbon-containing compounds.

[0032] In a sixteenth embodiment of the invention, which can be combined with all other embodiments, step (B) includes crystallization and / or extraction.

[0033] In the seventeenth embodiment of the invention, which can be combined with all other embodiments, the nitrogen-containing compound is selected from ammonia, ammonia water, (at least one) ammonium salt, soybean protein, urea, or a mixture of two or more of the above-mentioned nitrogen-containing compounds.

[0034] In the eighteenth embodiment of the invention, which can be combined with all other embodiments (except those excluding the following microorganisms), the microorganism in step (A) is selected from *Escherichia coli* (…). Escherichia coli ), Pseudomonas putida ( Pseudomonas putida ), Corynebacterium glutamicum ( Corynebacterium glutamicum Bacillus coagulans ( Bacillus coagulans (or a mixture of two or more of the above-mentioned microorganisms). Corynebacterium glutamicum is preferred. Particularly preferred... Corynebacterium glutamicum ATCC13032 .

[0035] In the nineteenth embodiment of the present invention, which is 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.

[0036] In the twentieth embodiment of the present invention, which is a specific 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.

[0037] In the twenty-first embodiment of the present invention, which is a specific embodiment of the eighteenth, nineteenth and twentieth embodiments, the fermentable carbon-containing compound is selected from starch hydrolysate, alkali metal salt of formic acid, ammonium formate, alkali metal salt of acetic acid, ammonium acetate, sugarcane juice, beet juice, hydrolysate of lignocellulose raw material, or a mixture of two or more of the above carbon-containing compounds.

[0038] In the twenty-second embodiment of the invention, which can be combined with all other embodiments (except those excluding the following microorganisms), the microorganism in step (A) is selected from *Assyriacus hymexazol* (…). Ashbya gossypii Pichia pastoris ( Pichia pastoris ), Hansenula polymorpha ( Hansenula polymorpha ), Max Kluyveromycin ( Kluyveromyces marxianus ), Yarrowia lipolytica ( Yarrowia lipolytica Bayer conjugated yeast ( Zygosaccharomyces bailii ), brewer's yeast ( Saccharomyces cerevisiae ), or a mixture of two or more of the above-mentioned microorganisms.

[0039] In the twenty-third embodiment of the present invention, which is the twenty-second embodiment, the reduction product is selected from formic acid or acetic acid.

[0040] In the twenty-fourth embodiment of the present invention, which is a specific 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.

[0041] In the twenty-fifth embodiment, which is a specific embodiment of the twenty-second, twenty-third, and twenty-fourth embodiments of the present invention, the fermentable carbon-containing compound is selected from starch hydrolysate, formic acid, acetic acid, sugarcane juice, beet juice, hydrolysate of lignocellulose raw material, or a mixture of two or more of the above-mentioned carbon-containing compounds.

[0042] In the twenty-sixth embodiment of the invention, which can be combined with all other embodiments (except those requiring a narrowing of the selection of microorganisms), the microorganism in step (A) is selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans, Ashurus spp., Pichia pastoris, Hansenula polymorpha, Kluyveromyces martensii, Yersinia lipolytica, Bayer conjugate yeast, Saccharomyces cerevisiae, or a mixture of two or more of the above microorganisms.

[0043] The following will To explain in more detailThe embodiments briefly described above, as well as other possible embodiments of the invention, are provided above. Unless those skilled in the art clearly see or explicitly state the contrary from the context, all the above embodiments and other embodiments of the invention described below can be combined with each other and in whole as needed.

[0044] Fermentation Step (A) of the method of the present invention involves Fermentation of fermentable carbonaceous compounds and... in the presence of microorganisms. Nitrogen compounds, to obtain aminobenzoate anions (H 2 NC 6 H 4 COO - ) and / or aminobenzoic acid (H 2 NC 6 H 4 COOH or H 3 N + C 6 H 4 COO - Fermentation broth Fermentation takes place in a reaction apparatus (i.e., a fermentation reactor) provided for this purpose. The reaction mixture present in the fermentation reactor is called... Fermentation liquid .

[0045] Fermentation in step (A) is preferably carried out in such a manner that the pH of the fermentation broth is in the range of 3.0 to 11. If necessary, the pH can be controlled by adding an alkali (particularly by adding ammonia water or gas, potassium hydroxide solution, or sodium hydroxide solution) (when the pH is too low) or by adding an acid solution (particularly hydrochloric acid, sulfuric acid, or nitric acid) (when the pH is too high). Different pH ranges within the aforementioned range may be particularly optimal for different microorganisms; this will be explained in detail below.

[0046] Preferred microorganisms for step (A) are prokaryotes (e.g., bacteria, in particular) or eukaryotes (e.g., yeast, in particular). Suitable microorganisms include, in particular, *Escherichia coli*, *Pseudomonas putida*, *Corynebacterium glutamicum*, *Bacillus coagulans*, *Ashurus spp.*, *Pichia pastoris*, *Hansenula polymorpha*, *Kluyveromyces martensii*, *Yersinia lipolytica*, *Zygosaccharizoa*, and *Saccharomyces cerevisiae*. Mixtures of different microorganisms may be used, but single-species microorganisms are preferred.

[0047] In principle, both approaches can be used to obtain this type of prokaryote or eukaryote, and these approaches can also be combined in preferred embodiments: (i) It can increase the enzymatic reactions in the aminobenzoic acid metabolic pathway in prokaryotic or eukaryotic cells, making the production of aminobenzoic acid faster than its consumption.

[0048] (ii) It can reduce or shut down subsequent reactions that convert aminobenzoic acid into other metabolites or products (e.g., tryptophan), resulting in even the rate of aminobenzoic acid formation being sufficient to achieve the enrichment of aminobenzoic acid in cells.

[0049] Methods for obtaining prokaryotes or eukaryotes with the aforementioned characteristics are known in the art. For example, suitable prokaryotes or eukaryotes can be identified by screening mutants that secrete aminobenzoic acid into the surrounding culture medium. However, it is preferable to use genetic engineering methods to specifically modify key enzymes. Using conventional genetic engineering methods, gene expression and enzyme activity can be enhanced, weakened, or even completely inhibited. Recombinant strains are such a result. For particularly preferred ortho-isomers, preferred embodiments are described below; applications to other isomers are within the ordinary capabilities of those skilled in the art. More preferably, prokaryotes or eukaryotes capable of converting fermentable carbonaceous compounds into aminobenzoic acid in the presence of nitrogen-containing compounds include modifications to the activity of an-aminobenzoic acid phosphoribosyltransferase, which reduce the enzyme activity. Due to this modification, an-aminobenzoic acid is converted into aminobenzoic acid... N The conversion of 5-(5-phosphate-D-ribosyl)-anaminobenzoic acid is reduced or completely inhibited. This leads to the accumulation of aminobenzoic acid in the cell. The expression "anaminobenzoic acid phosphoribosyltransferase activity" here refers to the catalytic conversion of 5-aminobenzoic acid to... N Enzymatic activity of α-(5-phosphate-D-ribosyl)-o-aminobenzoic acid.

[0050] In yeast, anthranilate phosphoribosyltransferase activity is genetically encoded by the native gene TRP4 (YDR354W). In the bacteria Corynebacterium glutamicum, anthranilate phosphoribosyltransferase activity is... trpD The genes (cg3361, Cgl3032, NCgl2929) encode it. For *Pseudomonas putida*, the encoding is through... trpDC within the manipulator trpD This was achieved through the gene (PP_0421).

[0051] In principle, the activity of the o-aminobenzoic acid phosphoribosyltransferase can be reduced in three ways: (i) Regulation of the expression of genes that can modify the activity of anthranilate phosphoribosyltransferase, thereby reducing or inhibiting the transcription or subsequent translation of such genes.

[0052] (ii) The nucleic acid sequence of the gene that enables anthranilate phosphoribosyltransferase activity can be modified so that the enzyme encoded by the modified gene has a lower specific activity.

[0053] (iii) The natural gene for anthranilate phosphoribosyltransferase activity can be replaced by a different gene derived from a different organism, wherein the different gene encodes an activity lower than that of the natural gene (e.g., TRP4, ...). trpD or trpDC The activity of anthranilate phosphoribosyltransferase (APPT) is that of the enzyme.

[0054] In a preferred embodiment of the invention, Corynebacterium glutamicum (preferably Corynebacterium glutamicum ATC13032) cells are used as microorganisms for providing a fermentation broth containing aminobenzoate anions. They preferably contain the modifications defined below. Further details regarding this strain and its metabolic capabilities are disclosed in WO 2023 / 111053.

[0055] (i) The activity of an-aminobenzoic acid phosphoribosyltransferase is reduced compared to the respective wild type. In one embodiment, there is no corresponding enzyme activity. Genetic modifications that enable this are well known. These modifications include deletion of the coding region of the relevant gene or the modified gene, such that the expressed enzyme is truncated or inactivated for some other reason. In an alternative embodiment, the enzyme activity has been reduced compared to the activity present in the wild type, although some residual activity must still exist. This residual activity is preferably between 10% and 60% of the natural activity in Corynebacterium glutamicum ATCC13032, more preferably between 20% and 50%. This is preferably achieved by reducing the activity of an-aminobenzoic acid phosphoribosyltransferase (...) compared to the wild type. trpD This is achieved by inhibiting gene expression, although expression is not completely suppressed. This is preferably done by using a promoter sequence with lower transcriptional activity compared to an endogenous promoter sequence, or by modifying the ribosome binding site to... trpD The distance of the start codon of a gene, or by changing the start codon itself. In a preferred embodiment of the invention, the activity of anthranilate phosphoribosyltransferase is reduced by: deletion or inactivation of endogenous anthranilate phosphoribosyltransferase (…). trpDThe gene is replaced with a gene for anthranilate phosphoribosyltransferase (e.g., as defined in SEQ ID NO. 1 or 2, preferably SEQ ID NO. 2) having a modified ribosome binding site and optionally a modified start codon. The amino acid sequence of the anthranilate phosphoribosyltransferase preferably corresponds to that of endogenous anthranilate phosphoribosyltransferase, more preferably as defined in SEQ ID NO. 3 or a variant thereof.

[0056] (ii) Increased activity of shikimate kinase. This is preferably achieved by increasing the expression of the corresponding enzyme. In one embodiment of the invention, the activity is increased by enhancing the expression of a gene for endogenous shikimate kinase as defined in SEQ ID NO. 6 or a variant thereof. In another preferred embodiment, this is achieved by expressing exogenous shikimate kinase (preferably as defined in SEQ ID NO. 7 or a variant thereof). Enhanced gene expression can be achieved by any method known to those skilled in the art, particularly by introducing two or more copies of the corresponding gene into a microorganism or by expressing the endogenous enzyme using a stronger promoter. A particularly preferred promoter for expressing exogenous genes or enhancing the expression of endogenous genes is P, as defined in SEQ ID NO. 8. tuf .

[0057] (iv) Enhanced activity of 3-phosphoshikimate 1-carboxyvinyltransferase and branched acid synthase. Preferably, these enzymes have amino acid sequences as defined in SEQ ID NO. 9 or variants thereof and SEQ ID NO. 10 or variants thereof. This is preferably achieved by introducing additional copies of the genes encoding these enzymes into the microorganism. tuf Promoters are preferably used to control expression.

[0058] (iv) The presence of 3-deoxyarabinohepenolate-7-phosphate synthase (DAHP synthase) is present, which is "feedback-resistant," meaning it is not inhibited by its product or any product formed from it. Preferably, the enzyme has the amino acid sequence defined in SEQ ID NO. 11 or a variant thereof.

[0059] Therefore, those skilled in the art will know that it is possible to introduce other modifications to improve the efficiency of the above strains based on the known metabolic pathway for the production of anthranilic acid in Corynebacterium glutamicum.

[0060] In another preferred embodiment of the invention, Escherichia coli (preferably Escherichia coli K12) cells are used as microorganisms for providing fermentation broth containing aminobenzoate anions, as disclosed in WO 2022 / 090363.

[0061] Escherichia coli strains expressing anthranilate phosphoribosyltransferase (TrpD) and glutamine aminotransferase (TrpG) are particularly preferred.

[0062] Preferably, the TrpG domain or a variant thereof is defined at amino acid sites 3 to 196 of SEQ ID NO. 13 (TrpGD from Escherichia coli). Also preferred are those derived from Bacillus subtilis (…). Bacillus subtilis TrpG (SEQ NO. 14) from Salmonella Typhimurium ( Salmonella typhimurium The TrpG domain of TrpGD (from amino acid sites 3 to 196 of SEQ ID NO. 15), TrpG from *Bacillus thuringiensis* (SEQ ID NO. 16), TrpG from *Corynebacterium glutamicum* (SEQ ID NO. 17), or a variant of any of the above polypeptides.

[0063] Preferably, the TrpD domain defined at amino acid sites 202 to 531 of SEQ ID NO. 13 (TrpGD from Escherichia coli) or a variant thereof is preferred. Also preferred are TrpD from Bacillus subtilis (SEQ NO. 18), the TrpD domain of TrpGD from Salmonella Typhimurium (at amino acid sites 202 to 531 of SEQ ID NO. 15), TrpD from Bacillus thuringiensis (SEQ ID NO. 19), TrpD from Corynebacterium glutamicum (SEQ ID NO. 20), or variants of any of the above polypeptides.

[0064] When TrpG and TrpD are expressed separately, it is preferable that the expression of TrpD is lower than that of TrpG. Preferably, it is at least 10% lower, more preferably at least 20%, even more preferably at least 40%, and most preferably at least 60%. However, it is particularly preferable to maintain the lowest possible expression of TrpD (at least 5% of the expression of TrpG). The expression of the genes encoding the above proteins is preferably determined at the mRNA level by quantitative TR-PCR. To achieve this differential expression of the two peptides, the genes encoding them are preferably introduced into cells in different expression cassettes (under the control of promoters of different strengths). Alternatively, different copy numbers of the respective genes may be introduced. In this case, the same promoter can be used for the expression of both peptides.

[0065] In yet another embodiment, cells of *Pseudomonas putida* (preferably *Pseudomonas putida* KT2440) are used as the microorganism for providing a fermentation broth containing an aminobenzoic acid anion. Genetic modifications enabling this bacterium to synthesize anthranilic acid are described in Example 4 of WO 2015 / 124687.

[0066] In this application, "variant" should be understood as a polypeptide obtained by adding, deleting, or substituting up to 20%, preferably up to 15%, more preferably up to 10%, and most preferably up to 5% of the amino acids present in a specific polypeptide. The above modifications can, in principle, be performed continuously or discontinuously at any desired site in the polypeptide. However, they are preferably performed only 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 acids have residues with similar chemical properties to those present in the unmodified polypeptide. Thus, amino acids with basic residues are more preferably exchanged with amino acids with basic residues, amino acids with acidic residues are exchanged with amino acids with similarly acidic residues, amino acids with polar residues are exchanged with amino acids with polar residues, and amino acids with nonpolar residues are exchanged with amino acids 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 unmodified polypeptide. Enzyme assays to verify the activity of the above enzymes can be found in the literature by those skilled in the art.

[0067] Aminobenzoic acid exists in three isomeric forms (o-aminobenzoic acid, m-aminobenzoic acid, and p-aminobenzoic acid). In principle, the method of the present invention can be applied to all three isomers, whether in isomerically pure form or as a mixture of different isomers. However, it is preferred to produce an-aminobenzoic acid or p-aminobenzoic acid, especially in isomerically pure form. Producing an-aminobenzoic acid, especially in isomerically pure form, is particularly preferred. In this respect, " Heterogeneous pure "In the terminology of this invention, it means that, based on all existing aminobenzoic acid isomers, the desired molar percentage of the aminobenzoic acid isomer is at least 99.0 mol%, preferably at least 99.9 mol%, more preferably 100 mol%. As is known in the art, the formation of the desired isomer can be controlled enzymatically. For example, in the shikimic acid pathway, the branched acid can be enzymatically converted to an-aminobenzoate (= the anion of an-aminobenzoic acid). Alternatively, there are also enzymatically catalyzed reactions that produce p-aminobenzoate (= the anion of p-aminobenzoic acid) from the branched acid."

[0068] Regardless of the microorganism used or the isomer required, the fermentation broth at the start of fermentation in step (A) contains recombinant cells of the microorganism used and at least one fermentable carbonaceous compound (and at least one nitrogenous compound as a nitrogen source). Preferably, the fermentation broth also contains other components selected from: buffer systems, inorganic nutrients, amino acids, vitamins, and other organic compounds required for the growth or housekeeping metabolism of the recombinant cells. The fermentation broth is water-based. Once fermentation has begun, the fermentation broth also contains the desired fermentation product, aminobenzoic acid (present in acidic or anionic form, depending on pH).

[0069] This document prefers fermentable carbon-containing compounds that can additionally serve as an energy and carbon source for the recombinant cell growth of the microorganisms used. Suitable for step (A) are, in particular, starch hydrolysates, formic acid, alkali metal salts of formic acid, ammonium formate, acetic acid, alkali metal salts of acetic acid, ammonium acetate, sugarcane juice, beet juice, hydrolysates of lignocellulose raw materials, and mixtures of two or more of the above-mentioned carbon-containing compounds. Acetic acid, formic acid, and their salts are preferably not used as the sole fermentable carbon-containing compound, but rather as a mixture with at least one of the other fermentable carbon-containing compounds.

[0070] Suitable nitrogen-containing compounds, especially ammonia, ammonia water, ammonium salts, soybean protein, urea, or mixtures of two or more of the above nitrogen-containing compounds.

[0071] As already mentioned, the pH value to be maintained during fermentation is determined by the microorganisms used. Microorganisms (e.g., *Escherichia coli*, *Pseudomonas putida*, *Corynebacterium glutamicum*, or *Bacillus coagulans*) are preferably cultured at a "neutral to alkaline pH" (i.e., particularly in the range of 5.5 to 11, preferably 6.0 to 8.0). It is preferred to use... Corynebacterium glutamicum, especially It is Corynebacterium glutamicum ATCC 13032 When fermentation is carried out at the above pH value, the aminobenzoic acid produced is mainly or even entirely its anion H2NC6H4COO. - 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. Regarding the fermentable carbonaceous compound in this embodiment, formic acid and acetic acid (if present) are preferably used not in their own form but in the form of their salts. Therefore, in the case of fermentation in a neutral to alkaline range, the fermentable carbonaceous compound particularly includes starch hydrolysate, alkali metal salts of formic acid, ammonium formate, alkali metal salts of acetic acid, ammonium acetate, sugarcane juice, beet juice, hydrolysate of lignocellulose raw materials, and mixtures of two or more of the above-mentioned carbonaceous compounds. Preferred are starch hydrolysate, sugarcane juice, beet juice, and / or hydrolysate of lignocellulose raw materials.

[0072] In contrast, the microorganisms (e.g., *Ashurus spp.*, *Pichia pastoris*, *Hansenula polymorpha*, *Kluyveromyces martensii*, *Yersinia lipolytica*, *Bayer's conjugate yeast*, or *Saccharomyces cerevisiae*) are preferably cultured in an acidic environment (i.e., particularly at a pH range of 3.0 to <5.5, preferably 3.5 to 5.0). Depending on the specific pH range of 3.0 to <5.5, the aminobenzoic acid in the fermentation broth is in anionic form (H₂NC₆H₄COO). - ) or in electrically neutral form (H2NC6H4COOH or H3N + C6H4COO -(At the lowest pH value within the above range, i.e., pH 3.0 or slightly above 3.0, the cation H3N cannot be completely eliminated.) + The formation of C6H4COOH, but which accounts for at most 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. Regarding the fermentable carbonaceous compounds in this embodiment, formic acid and acetic acid (if present) are preferably used in their own form, i.e., preferably not in the form of salts. Therefore, in the case of fermentation in an acidic range, the fermentable carbonaceous compounds particularly include starch hydrolysates, formic acid, acetic acid, sugarcane juice, beet juice, hydrolysates of lignocellulose raw materials, and mixtures of two or more of the above-mentioned carbonaceous compounds. Preferred are starch hydrolysates, sugarcane juice, beet juice, and / or hydrolysates of lignocellulose raw materials.

[0073] In one embodiment of the invention, step (A) is performed continuously, i.e., the reactants are continuously fed into the fermentation reactor and the product is continuously removed from the fermentation reactor. In the simplest case, the product continuously removed from the fermentation reactor is a fermentation broth containing microorganisms present therein and containing aminobenzoate anions and / or aminobenzoic acid. However, it is also contemplated to use known separation methods (especially filtration) to retain and remove the microorganisms from the fermentation reactor. Clarification Fermentation broth containing aminobenzoate anions and / or aminobenzoic acid. Of course, this clarification of the fermentation broth can also be carried out outside the fermentation reactor, particularly by filtration, centrifugation, or sedimentation.

[0074] In another embodiment of the invention, step (A) is carried out in a discontinuous operation (referred to as "batch mode") during the fermentation cycle. The fermentation cycle preferably includes initial loading or addition of microorganisms to the culture medium, initial loading and / or addition of nutrients, accumulation of microorganisms, formation of the desired product (i.e., aminobenzoic acid), and complete or partial evacuation of the reactor after fermentation. In a variant of the batch mode (referred to as "feed-batch mode"), reactants are fed (continuously or discontinuously [i.e., in batches]) into the fermentation reactor as long as the reactor volume allows, without removing the product (except for any gaseous components discharged to the exhaust system through the fermentation reactor connector). The reaction is stopped after adding the maximum possible amount of reactants, and the product mixture is removed from the fermentation reactor. In the discontinuous operation mode, it is preferable to clarify the fermentation broth outside the fermentation reactor, particularly by filtration, centrifugation, or sedimentation.

[0075] The removed microorganisms (biomass) can be recycled back into the fermentation process.

[0076] Regardless of the specific operating mode, the fermentation reactor preferably includes means for measuring important process parameters, such as temperature, pH, substrate and product concentrations, dissolved oxygen content, and cell density of the fermentation broth. Particularly preferably, the fermentation reactor includes means for adjusting at least one (preferably all) of the above-mentioned process parameters.

[0077] Suitable fermentation reactors are stirred tank reactors, membrane reactors, or loop reactors. Stirred tank reactors and loop reactors are particularly preferred for both aerobic and anaerobic fermentation (airlift reactors are preferred, as they circulate the liquid in the reactor through bubbling).

[0078] Post-processing of fermentation broth (workup) Step (B) of the method of the present invention relates to Aminobenzoic acid obtained from fermentation broth .

[0079] Step (B) is performed based on the pH of the fermentation. If it is sufficiently higher than the isoelectric point of the aminobenzoic acid isomer to be obtained (pH 3.50 for o-aminobenzoic acid), then aminobenzoic acid will be predominantly or entirely in anionic form, and thus in dissolved form. In this case, it is preferable to first clarify the fermentation broth, i.e., remove the suspended solids (e.g., especially the microorganisms used), thereby obtaining a clarified fermentation broth containing the dissolved aminobenzoic acid anion. Conventional methods suitable for clarification are used, such as filtration, centrifugation, or sedimentation. The aminobenzoic acid can then be obtained from the clarified fermentation broth by methods known in the art and explained in more detail below.

[0080] If the pH of the fermentation is close to or significantly lower than the isoelectric point of the aminobenzoic acid isomer to be obtained, then at least a large portion of the aminobenzoic acid will be in the form of an acid (rather than its anionic form). Due to the low water solubility of aminobenzoic acid, it will subsequently exist in the fermentation broth in suspension, at least to a large extent, as a solid. Here, the operation can also be to first clarify the fermentation broth as described above, and then obtain the amount of aminobenzoic acid or its anions dissolved in the clarified fermentation broth by methods known in the art. However, since most of the aminobenzoic acid has already been obtained in solid form along with the microorganism, the microorganism must be removed from the solid. This can be done, for example, by treating (extracting) the solid with an aqueous alkaline solution (e.g., sodium hydroxide or potassium hydroxide) or an aqueous solution of a strong acid (hydrochloric acid or sulfuric acid), thereby converting the aminobenzoic acid into its anionic or cation and selectively dissolving it. This is followed by solid-liquid separation using conventional methods (e.g., particularly filtration, centrifugation, or sedimentation). Aminobenzoic acid can then be obtained from the resulting aqueous solution containing aminobenzoate anions or protonated aminobenzoic acid by methods known in the prior art (crystallization by adjusting the pH to a value equal to or close to the isoelectric point). It is also conceivable to directly treat the fermentation broth with an alkaline or acidic aqueous solution without prior clarification.

[0081] Alternatively, aminobenzoic acid present in the solid obtained from the clarification of the fermentation broth can also be extracted with an organic solvent. It is also conceivable to extract the fermentation broth directly with an organic solvent without prior clarification. Suitable organic solvents are, for example, C8-C4. 12 Alkanols or anilines (especially the latter, if aminobenzoic acid is only an intermediate in the production of aniline). Aminobenzoic acid can be readily obtained by evaporation and concentration of the resulting solution. Depending on the intended subsequent application, the resulting aminobenzoic acid solution can also be further processed directly in an organic solvent (e.g., decarboxylation); such steps are also covered in the wording. From the fermentation broth get aminobenzoic acid middle.

[0082] In any case, the preferred operating procedure is to concentrate the resulting solution (in an alkaline aqueous solution or an organic solvent) as much as possible.

[0083] The extraction of aminobenzoic acid from an aqueous solution (clear fermentation broth or alkaline extract) of the aminobenzoic acid anion is preferably achieved by crystallization. Depending on the available concentration, it may be useful to remove a portion of the water present in this step to facilitate the separation of aminobenzoic acid. Removal can be achieved by techniques known in the art, particularly by evaporation or by membrane method. All evaporation equipment known in the field is suitable for evaporation. To minimize heat load, evaporation is preferably carried out under reduced pressure, particularly at 0.1 mbar to 900 mbar, more preferably at 100 mbar to 500 mbar. This allows for the gentle evaporation of water at temperatures from 45°C to 97°C (particularly up to 82°C).

[0084] In this specialized field, suitable technical equipment (as a crystallizer) is used for crystallization. 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 a value ranging from 3.0 to 4.7, preferably 3.2 to 3.7, more preferably 3.4 to 3.6, and most preferably 3.5. This is preferably accomplished by adding an acid selected from hydrochloric acid, sulfuric acid, or phosphoric acid. This pH adjustment results in the formation of aminobenzoic acid anions (H₂NC₆H₄COO₃). - It is mainly or even completely converted into its electroneutrally neutral form (H2NC6H4COOH or H3N). + C6H4COO - Aminobenzoic acid and its crystals. This type of crystallization is also called reaction crystallization. Crystallized aminobenzoic acid can be separated by known methods (e.g., in particular filtration, sedimentation or centrifugation), leaving an aqueous mother liquor.

[0085] It has been found useful to feed the fermentation broth and acid into the crystallizer via spatially separated feed devices (as far apart as possible). This achieves the effect of thorough mixing of the reactants with the reactor contents before the acid-base reaction occurs. Examples of useful feed devices include pipes, preferably equipped with barrier valves. In one embodiment, the feed device for the fermentation broth and the feed device for the acid are arranged at opposite sites on the reactor wall, (substantially) perpendicular to it. In another embodiment, the feed device for the fermentation broth and the feed device for the acid are arranged (substantially) parallel to the reactor wall, wherein the feed devices are opposite each other and as close as possible to each other on the reactor wall, particularly directly opposite each other.

[0086] The crystallizer can be divided into chambers using suitable internal components. The flow direction can be adjusted by selecting the agitator geometry and operating mode. An external pumping circulation system can also be provided for the crystallizer, in which case one of the two reactants (fermentation broth or acid) is introduced into the pumping circulation system, while the other is introduced directly into the crystallizer. If the crystallizer is operated with a sifter and a pumping circulation system, the pumping circulation system is used at the bottom of the sifter for agitation or on the side of the sifter.

[0087] Crystallization in the crystallizer can be carried out continuously or in batches. Continuous operation is preferred. Regardless of the operating mode (continuous or batch), the exact operating parameters (among others) are determined by the desired crystal size, which can be adjusted by residence time / reaction time and supersaturation level (longer residence time / reaction time and lower supersaturation level promote larger crystal size).

[0088] Crystallization is preferably carried out in the presence of seed crystals: In batch crystallization, a preferred operating procedure is to first fill the fermentation broth into a crystallizer and adjust it to a defined temperature (preferably 5°C to 40°C, e.g., 20°C). If the pH of the fermentation broth at the selected temperature is significantly higher than the pH at which the minimum solubility level of aminobenzoic acid is reached, the clarified fermentation product is initially only slightly acidified, specifically to a pH that has not yet reached the minimum solubility level of aminobenzoic acid under the selected temperature and given boundary conditions, but is significantly closer to that pH (preferably acidified to pH 5.0 to 6.5 in this first step). This slight acidification can be achieved quickly. Seed crystals of the desired aminobenzoic acid polymorph are then added; in the case of o-aminobenzoic acid, this is preferably polymorph (form) I. This polymorph has relatively low solubility and thus facilitates a very large recovery of aminobenzoic acid. The amount of seed crystals added is preferably about 0.1% to 1% of the aminobenzoic acid dissolved in the fermentation broth. In this way, a suspension of seed crystals is obtained (see also WO 2017 / 085170 A1). Subsequently, the pH is adjusted to 3.0 to 4.7, preferably 3.2 to 3.7, more preferably 3.4 to 3.6, and most preferably 3.5, by adding acid (the same acid used in the previous slight acidification). The acid is preferably added gradually; for example, over 1 hour, with 1 kg of the initially loaded fermentation broth and using 37% hydrochloric acid. After the acid addition is complete, stirring continues for a certain period, particularly the same period as the time used for adding acid after adding the seed crystals. The precipitated aminobenzoic acid is then separated by filtration (under vacuum if necessary), sedimentation, or centrifugation (preferably by centrifugation), and preferably by washing once or more (particularly twice) with an acidic water wash (particularly the same acid used for precipitation) at a pH of 3.0 to 4.7, preferably 3.2 to 3.7, more preferably 3.4 to 3.6, and most preferably 3.5. Depending on the purity required for the intended subsequent application, the aminobenzoic acid can also be purified by recrystallization.

[0089] In continuous crystallization, seed crystals are typically added only at the start-up stage of the continuous process, as more seed crystals will spontaneously form in situ subsequently (a process known as secondary nucleation). The seed suspension required for startup can be provided as described above for batch crystallization. Post-treatment (removal and washing of the crystallized aminobenzoic acid) can also be performed as described above.

[0090] In addition to clarification, the fermentation broth can undergo other pretreatment steps before crystallization. Particular attention should be paid to the decolorization of the fermentation broth (especially the already clarified broth). This decolorization is preferably carried out in such a manner that the (preferably clarified) fermentation broth is passed through a column packed with solid packing material to remove the dye by adsorption. Possible solid phases that can be used are, for example, diatomaceous earth or ion exchange packing material. This decolorization is preferably carried out when the fermentation broth contains colored substances that can interfere with subsequent crystallization in step (B).

[0091] The conversion of carbon dioxide into reduction products Step (C) of the method of the present invention includes Carbon dioxide is converted into an alkali metal selected from formic acid, ammonium formate, and formic acid. Reduction products of salts, acetic acid, ammonium acetate, or alkali metal salts of acetic acid .

[0092] In principle, the carbon dioxide used in step (C) can come from any source, provided that it is present in it at the purity required for reduction or that such purity can be achieved through an acceptable effort.

[0093] Preferred sources of carbon dioxide are those inherent to the process, in this context Inherent source of technology This should be understood to specifically refer to the further processing of aminobenzoic acid to generate its direct reaction product and simultaneously form carbon dioxide. Using the carbon dioxide generated during fermentation in step (A) is technically possible, but not preferred, because this carbon dioxide is produced in a highly diluted form, and its concentration would be extremely expensive and inconvenient.

[0094] Carbon dioxide from the further processing of p-aminobenzoic acid to produce its direct reaction products, particularly carbon dioxide produced during the decarboxylation of aminobenzoic acid to aniline (see below for more details). This carbon dioxide from decarboxylation is particularly suitable because the impurities present are at most trace amounts of aniline or o-aminobenzoic acid, which are easily removed.

[0095] As already mentioned, the carbon dioxide used for reduction can also be from an external source. A combination of process-specific and external sources can also be used, meaning the carbon dioxide used can be a mixture of carbon dioxide from a process-specific source (e.g., particularly decarboxylation) and carbon dioxide from an external source. Several sources are conceivable as external sources, such as carbon dioxide from the following: • Processes used to produce (i) bioethanol, (ii) cement, (iii) hydrogen (from steam reforming, for example, including CO conversion or microbial electrolysis[3]), or (iv) epoxides, or • Biodegradation processes that take place in digesters (especially in wastewater treatment plants or biogas plants), or • From fuel combustion, or • The process used to extract carbon dioxide from the air (so-called Direct air capture ).

[0096] (Regarding [3], see: Abudukeremu Kadie) 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 method of the present invention, the extraction of carbon dioxide from an external carbon dioxide source is achieved by adsorption, absorption, and / or membrane methods known in the art. Such methods are also suitable for extracting carbon dioxide from fermentation waste gases.

[0097] For example, pressure swing adsorption (PSA) on activated carbon, molecular sieves, and carbon molecular sieves can be used to remove carbon dioxide from gas mixtures. This is based on the difference in adsorption behavior of the adsorbent (the adsorbate, i.e., the gaseous components bound to it, in both solid and stationary phases) on the gaseous components. Amine washing can also be used. This is an absorption method where the chemical reaction between the gaseous components and the solution overlaps with physical absorption, and it can absorb much more carbon dioxide. The washing agent used is an amine solution. Membrane methods use diffusion membranes, which utilize the differences in the solubility of gaseous components within the membrane.

[0098] Removing carbon dioxide from ambient air (the so-called Direct air capture (Or DAC) can also be used to provide carbon dioxide for further processing in the methods of this invention. The most important DAC methods employed to date can be broadly subdivided into the following three technical paths: Absorption and electrodialysis are methods in which carbon dioxide present in the inhaled air is absorbed using a sodium hydroxide solution. The resulting sodium carbonate solution is acidified with sulfuric acid, allowing the carbon dioxide to be separated again in a nearly pure form. This is then followed by the recovery of the sodium hydroxide solution and sulfuric acid via an electrochemically driven membrane process (electrodialysis).

[0099] In the case of absorption and calcination methods, as described above, carbon dioxide is absorbed using an alkali metal hydroxide solution (sodium hydroxide or potassium hydroxide solution). When using potassium hydroxide solution, the potassium carbonate (hydrogen carbonate) solution produced by carbon dioxide absorption is precipitated in a pellet reactor to form calcium carbonate, which is then decomposed into carbon dioxide and calcium oxide by calcination. The latter is hydrated to form calcium hydroxide, which can then be used in the next cycle.

[0100] In the case of adsorption and desorption methods, carbon dioxide is first bound to the adsorbent via organic chemical adsorption, and then the adsorbent is regenerated, particularly by providing heat or moisture. The filter material used can be, in particular, dried cellulose with amine compounds attached to its surface or resin with amines attached.

[0101] The actual conversion of carbon dioxide into reduction products is preferably achieved by reaction with hydrogen or by electrolysis, the methods of which are known in the art and are therefore only briefly reported below.

[0102] The reduction of carbon dioxide to formic acid or its salts using hydrogen is achieved using suitable catalysts, particularly ruthenium and iridium catalysts with nitrogen-containing and / or phosphorus-containing ligands. Suitable methods are described in, for example, Chem. Rev. 2015, 115 , 12936-12973 by W.-H. Wang, Y Himeda, JT Muckerman, GF Manbeck andE. Fujita ( "CO 2 Hydrogenation to Formate and Methanol as an Alternative to Photo- and Electrochemical CO 2 Reduction” See, for example, Scheme 2 (formic acid) and Scheme 3 (formate salt) in [4].

[0103] Using ruthenium catalysts (e.g., especially [Ru(H)2(P)2) n Bu3)4]), a method for obtaining formic acid by hydrogenation of CO2 using an amine (e.g., particularly trihexylamine) and a polar solvent (capable of forming hydrogen bonds) (e.g., a diol, particularly 2-methyl-1,3-propanediol, 1,3-propanediol, 1,2-propanediol, or ethylene glycol) is described in T. Schaub and RA Paciello in Angew. Chem. 2011, 123 , 7416 –7420 ( "Ein Verfahren zur Herstellung von Ameisensäure durch CO 2 -Hydrierung: Thermodynamik und die Rolle von CO” [5] The disclosed concept is a multiphase liquid-liquid process that allows for the recovery of amines, polar solvents and catalysts and the removal of formic acid from the system by distillation.

[0104] The reaction of carbon dioxide with hydrogen to form formic acid can also be achieved through biotechnological methods; see, for example, F. Oswaldet. al. , ( "Formic acid Formation by Clostridium ljungdahlii at elevated Pressures of carbon Dioxide and hydrogen” ), Front. Bioeng. Biotechnol. 2018, Volume 6, Article 6 [6].

[0105] The electrochemical reduction of carbon dioxide to formic acid is achieved through acidic electrolysis, specifically at a pK value lower than that of formic acid. a (3.77) pH value, particularly at pH 2.00 to 3.75. Electrolytes suitable for this purpose and operating modes are described by M. Oßkopp, A. Löwe, CMS Lobo, S. Baranyai, T. Khoza, M. Auinger and E. Klemmin. Journal of CO2 Utilization 2022, 56 , 101823 ( "Producing formic acid at low pH values by electrochemical CO 2 reduction” ) [7] in.

[0106] The electrochemical reduction of carbon dioxide to formate occurs correspondingly at pK values ​​higher than those of formate. a This is achieved at a suitable pH (e.g., at pH 10). The suitable electrolyzer and operating mode for this purpose are described in 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]

[0107] The electrochemical reduction of carbon dioxide to acetic acid is described in Ratnadip De et al. in Angew. Chem. Int. Ed. 2020, 59 , 10527 – 10534 ( "Electrocatalytic Reduction of CO 2 to Acetic Acid by a Molecular Manganese Corrole Complex” [9] In. Another conceivable approach is to reduce CO2 to methanol (particularly by catalytic hydrogenation), and its use in the Monsanto process for producing acetic acid by carbonylating methanol with carbon monoxide. The carbon monoxide required for carbonylation can also be obtained by catalytic hydrogenation or electrochemical reduction (see, for example, WO 2021 / 069498 A1).

[0108] Acetates can be obtained by reacting CO2 or a mixture of CO2 and CO with hydrogen in a biotechnological process; see the aforementioned publications. Microbial Biotechnology 2018, 11 (4), 606 – 625 [1], for example, Figure 2. Electrochemical methods also exist; see, for example, HH Heenen et al., "The mechanism for acetate formation in electrochemical CO (2) reduction on Cu: selectivity with potential, pH, and nanostructuring” , published in Energy Environ. Sci. , 2022, 15 , 3978 – 3990

[10] .

[0109] Adding reduction products to fermentation Step (D) of the method of the present invention relates to The reduction product from step (C) is added to the fermentation in step (A). middle The recycled reduction product is used as a carbon source for the microorganisms in fermentation, thereby reducing the need for primary fermentation substrates (e.g., sugars) (see also, for example, an exemplary embodiment involving the use of acetate). If fermentation is carried out at a pH where aminobenzoic acid is produced primarily or entirely as an aminobenzoate anion (e.g., as sodium aminobenzoate), and the reduction product used is a (particularly alkali metal) formate or acetate, this has the additional advantage of the formate or acetate counterion being available as the counterion of the fermentation product aminobenzoate anion. In this way, pH is stabilized, and the need for additional base (e.g., sodium hydroxide solution) for pH adjustment is reduced or completely eliminated.

[0110] Therefore, this ultimately results in the following advantages: (1) lower production costs because of lower consumption of substrate and base in the production of the target product; (2) a lower CO2 footprint of the target product aminobenzoic acid because fewer raw materials (e.g., sugar and base) are required (in addition, in the case of further processing to produce aniline, it is possible to recycle the CO2 formed in the decarboxylation and supplement it with CO2 from other sources if necessary, thus avoiding its emissions).

[0111] To illustrate the advantage of substrate economy, the effect of the supply of reduction products on the theoretical yield was calculated. The theoretical yield is defined in each case as the maximum mass [g] of anthranilic acid achievable per unit mass [g] of glucose or per unit mass [g] of organic acid (if glucose is not used). It is calculated by establishing elemental balance (balance of carbon, nitrogen, and hydrogen atoms) and charge balance (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 used are glucose and a specific organic acid (alone or in a specified molar ratio), as well as ammonia and molecular oxygen. Permitted products are anthranilic acid, water and carbon dioxide, with the amount of anthranilic acid maximized and the formation of water and carbon dioxide used to balance excess hydrogen and oxygen. Regarding the use of glucose and formic acid in a 1:1 molar ratio (see also input 4 in Table 1 below), the reaction equation is as follows: 14 C6H 12 O6 + 14 CHOOH + 13 NH3 13 H2N(C6H4)COOH + 72 H2O + 7 CO2.

[0112] Metabolic pathways and thermodynamics were not considered in this calculation. The results are summarized in Table 1.

[0113] Table 1: Effect of the method of the present invention on the yield of o-aminobenzoic acid (oAB) Symbol explanation: pa = operating steps in the prior art; ref. = reference; inv. = operating steps of the present invention. As shown in Table 1, the amount of glucose can be reduced in the operating steps of the present invention without considering a decrease in yield.

[0114] The reduction product can be added to the fermentation continuously or in batches. It should be noted that the reduction product is added in such a manner that the concentration of the reduction product in the fermentation broth (used as an additional carbon source for the microorganisms; see above) is kept below a concentration that would be toxic to the microorganisms. This limit varies depending on the microorganisms and the type of reduction product supplied, and can be easily determined by a preliminary test.

[0115] For further information on microbial utilization of formate, see Justine Turlin. et al. , Metabolic 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 A1.

[0116] Uses of aminobenzoic acid in the production of other valuable products After any necessary further purification using known methods (e.g., recrystallization), the aminobenzoic acid obtained in step (B) is suitable for all applications of aminobenzoic acid known in the art.

[0117] For example, anthranilic acid is an important starting material for the synthesis of anthranilic esters (an important fragrance), indigo, pharmaceuticals, and crop protectants (acaricides). The anthranilic acid produced by this invention is suitable for all these purposes.

[0118] The aminobenzoic acid produced by this invention is preferably used in the production of polymers. In this respect, the method of this invention can make an important contribution to more sustainable plastics production, which often requires large-scale production. For example, anthranilic acid can be converted into poly(anthranilamide), as described in WO 2022 / 008449 A1 (after conversion to indocyanine anhydride) or WO2022 / 008450 A1 (after conversion to anthranilic ester). As described in WO 2022 / 122906 A1, the anthranilic acid obtained by this invention can also be converted into anthranilic acid derivatives selected from anthranilyl halides, indocyanine anhydride, or mixtures thereof, and then reacted with polyols to form polyamines, which are then suitable for phosgenation to form the corresponding polyisocyanates.

[0119] Specifically, the aminobenzoic acid obtained by this invention can also be decarboxylated to aniline, which is a particularly important raw material in the polyurethane industry.

[0120] Aniline obtained in this manner can then be provided for all known uses. Specifically, these include a (acid-catalyzed) reaction with formaldehyde to form methylene diphenylene diamine and polymethylene polyphenylene polyamine, which is then phosgenated to form methylene diphenylene diisocyanate and / or polymethylene polyphenylene polyisocyanate, followed by a reaction with a polyol to form a polyurethane. It goes without saying that aniline obtained by decarboxylating aminobenzoic acid produced in this invention can also be provided for various uses, such as the production of azo compounds. For example, this includes the production of methyl red, which is achieved by diazotizing the amino group of o-aminobenzoic acid with sodium nitrite and hydrochloric acid, followed by reaction with… N , N Obtained by dimethylaniline azo coupling.

[0121] Decarboxylation of aminobenzoic acid As already mentioned, the method of the present invention includes, in a particularly preferred embodiment, the decarboxylation of aminobenzoic acid to aniline.

[0122] Decarboxylation can be achieved as is generally known in the prior art. A catalyst can be used, but it is not necessarily required.

[0123] Examples of suitable catalysts include aqueous acids such as sulfuric acid, nitric acid, and hydrochloric acid; solid acids such as zeolites and Si-Ti molecular sieves; solid bases such as hydroxyapatite and hydrotalcite; and polymeric acids such as ion exchange resins (preferably Amberlyst). Particularly preferred catalysts are described in WO 2022 / 253890 A1. The catalysts disclosed therein are characterized by a high mass proportion of alumina (at least 40%). The alumina is preferably γ-Al₂O₃ or η-Al₂O₃, particularly when no other metal oxides are present. In principle, other metal oxides may also be present in addition to alumina, particularly containing magnesium oxide (MgO), with a mass proportion (based on the total mass of the metal oxides) of 1.0% to 60.0%, preferably 2.0% to 50.0%, and particularly preferably 5.0% to 35.0%. In addition, the catalyst may contain SiO2 in a mass ratio (based on its total mass) of 1.0% to 30.0%, preferably 2.0% to 20.0%, more preferably 2.0% to 10.0%.

[0124] Therefore, in terms of reaction conditions, 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 in the range of 180°C to 240°C. The (absolute) reaction pressure can be from 0.05 bar to 300 bar, preferably 1.0 bar to 100 bar, and more preferably 1.0 bar to 60 bar.

[0125] Decarboxylation can also be carried out in the presence of aniline, i.e., aminobenzoic acid dissolved in aniline. Since aniline catalyzes the decarboxylation of aminobenzoic acid, thereby accelerating its formation (autocatalysis), the use of a system-extraneous catalyst is not absolutely necessary in this variant; see also WO 2020 / 020919A1, which describes this method. Unlike the steps disclosed in WO 2020 / 020919 A1, purified aniline can also be used as a solvent for aminobenzoic acid. Furthermore, unlike the steps disclosed in WO 2020 / 020919 A1, an additional system-extraneous catalyst can be used. In the case of a batch reaction, during decarboxylation... Before starting Preferably, the mass percentage of aniline (based on the total mass of aniline and aminobenzoic acid) is determined to be from 0.1% to 90%, more preferably from 1.0% to 70%, and particularly preferably from 5.0% to 50%. In the case of a continuous reaction, the mass percentage of aniline (based on the total mass of aniline and aminobenzoic acid) is continuously determined to be from 0.1% to 90%, more preferably from 1.0% to 70%, and particularly preferably from 5.0% to 50% during the decarboxylation process.

[0126] Of course, other solvents or diluents besides aniline can also be used, especially water. Other suitable preferred solvents are organic, polar or protic solvents, such as halogenated aliphatic or aromatic hydrocarbons, straight-chain or cyclic ethers, straight-chain or cyclic esters, straight-chain or cyclic amides, alcohols, ketones, nitriles, phenol derivatives, benzanilide, sulfonamides or sulfolane, preferably having a boiling point above the selected reaction temperature under the selected conditions, and preferably forming a homogeneous reaction mixture with the reactants at that temperature.

[0127] Regarding the reaction method, both gas and liquid phases are suitable. The reaction can be carried out continuously (preferably) or in batches.

[0128] Preferred operating steps include decarboxylating aminobenzoic acid. • It is carried out in a reactor (especially a tubular reactor) in the liquid or gas phase, the reactor having an integrated fixed catalyst bed (comprising a catalyst bed as a shaped body (extrusion) or a catalyst comprising an integral structural form). • Carrying out in a fluidized bed reactor in the liquid phase or preferably the gas phase, or • A suspension containing a catalyst ( slurry The process is carried out in the liquid phase in a stirred tank.

[0129] In this article, tubular reactor This should be understood as referring to a tubular reactor, in the case of a continuous reaction (preferably), through which the reaction mixture flows during operation. Tubular reactors with a small length-to-diameter ratio are also called... Tower reactor They are also covered in the term "tubular reactor" as understood in this article.

[0130] Using molded catalyst bodies (extrusions) or monolithic catalyst structures allows for easy reuse of the catalyst once decarboxylation is complete.

[0131] The catalyst present at the time of decarboxylation completion is preferably regenerated before reuse. This can be done by washing the catalyst with an organic solvent or aqueous solution, and / or by completely burning it out at high temperature in the presence of O2 to remove organic deposits.

[0132] The aniline formed can be separated and purified using standard techniques (especially distillation) and used as described above.

[0133] Example Effect of potassium acetate addition on the yield of anthranilic acid Description of the strain All experiments were conducted using the strains described below.

[0134] Starting with the bacterium Corynebacterium glutamicum ATC13032, targeted chromosome modification was performed to generate a microbial strain that produces anthranilic acid. All genetic modifications (i.e., chromosome deletions and gene integrations) were performed using the corresponding pK19. mobsacB The derivatives are achieved through di-homologous recombination (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 1994, 145 (1), 69 – 73

[13] ; doi: 10.1016 / 0378-1119(94)90324-7).

[0135] By first lacking natural trpD Alleles were replaced with alleles that have a GTG start codon instead of an ATG start codon and a ribosome binding site with a shortened distance from the start codon (called...). trpD5 (SEQ ID NO. 1) to reduce the activity of anthranilate phosphoribosyltransferase TrpD.

[0136] The gene encoding one or only phosphoenolpyruvate carboxylase (SEQ ID NO. 4) in Corynebacterium glutamicum was deleted (SEQ ID NO. 5).

[0137] To promote aromatic biosynthetic pathways, artificial polycistronic P tuf - aroLAC The operator (SEQ ID NO.12) is integrated into cg2563 Downstream, this operon is derived from E. coli aroL ( b0388 ) genes and from Corynebacterium glutamicum aroA ( cg0873 )and aroC ( cg1829 The gene composition is controlled by the constitutive promoter of the elongation factor Tuf. Furthermore, aroG encodes an anti-feedback variant (SEQ ID NO. 11) of the DAHP synthase from *E. coli*. new Alleles integrated into the strain genome cg3132 Downstream of.

[0138] Operating steps Comparative cultures of the above-mentioned strains used for the production of anthranilic acid were conducted starting with 20 g / L D-glucose and different concentrations of potassium acetate.

[0139] Table 2:Overview of the concentrations of the carbon sources D-glucose and potassium acetate in the main culture medium. For media 1 to 4, two replicates were set up for each case, with an initial culture volume of 50 mL for each. For each condition from Table 2, two replicates were set up for each case. The culture conditions are shown below (Table 3).

[0140] Table 3: Overview of pre-culture and master culture conditions. Both secondary seed culture and master culture (initial 50 mL in each case before the first sampling) were performed in Erlenmeyer flasks with a maximum volume of 1000 mL. The sterile barrier used was cotton plugs. The culture medium used, its composition, and its preparation Unless otherwise stated, all culture media were prepared with ultrapure water (MilliQ H2O) and autoclaved.

[0141] Table 4: Used for cell growth Brain and heart immersion fluid A liquid and solid composite culture medium consisting of Brain Heart Infusion (BHI). Table 5: A liquid basic culture medium with a complex composition used for cell growth in seed culture. The measured amount is used for 1 L of complete CGXII seed medium. Complete replenishment is required to reach the final desired concentration in the complete medium. MOPS = (3-( N -morpholino)propanesulfonic acid) Table 6: Liquid basic medium for the main culture. The measured amount is used for 1 L of complete CGXII main medium. Complete replenishment is required to reach the final desired concentration in the complete medium. Table 7: Overview of the preparation of a 2 g / L biotin stock solution. The solution was sterilely filtered (0.2 µm) rather than autoclaved. This solution can be stored at 4°C for 1 month. Table 8: Overview of the preparation of 600 g / L D-glucose stock solution. Table 9: Overview of the preparation of 327 g / L potassium acetate stock solution. Table 10: Overview of the preparation of 10 g / L CaCl2 stock solution. Table 11: An overview of the preparation of 200 g / L MgSO4 stock solution. Table 12: Overview of the preparation of trace element stock solutions. The solution is sterilely filtered (0.2 µm) rather than autoclaved. The solution has a shelf life of 6 months at 4°C. Due to the small volume produced, it is recommended to prepare 1 L per batch. Table 13: Overview of the preparation of 1× phosphate-buffered saline (PBS). 10× PBS (catalog number BP399-1) from Fisher Scientific GmbH was used. The instruments used Table 14: An overview of the parameters examined in this study and the instruments and methods used for the examination. Operating steps Starter cultures were generated by extracting cell masses from glycerol-dominant microbial cultures used for culturing and inoculating them onto BHI agar plates. The BHI agar plates were incubated overnight at 30°C.

[0142] The agar plate cultures established in this manner were used to inoculate BHI liquid cultures. For this purpose, a small number of cell clusters were removed from each BHI agar plate culture, and 4.5 mL of liquid BHI medium was inoculated into round-bottom test tubes in each case. The liquid cultures were incubated at 30°C and 200 rpm for 7.5 h (preculture I).

[0143] For secondary seed culture, CGXII seed medium containing 20 g / L D-glucose was used. To do this, two shake flasks (each containing 49 mL of seed medium) were transferred to a 1000 mL Erlenmeyer flask, 1 mL of BHI liquid seed culture was added, and the two shake flask seed cultures were incubated at 200 rpm and 30 °C for 17 h.

[0144] Seed culture II was used to inoculate the master culture. For this purpose, after determining the optical density, both cultures were first completely centrifuged, and the resulting cell pellets were resuspended in PBS buffer. The optical density was set to 50. The suspensions were combined, and 1 mL of the same cell suspension was used to inoculate all master cultures in each case, setting the initial optical density (OD) to 50. 600 The target value was 1. Two cultures were prepared for each master culture condition.

[0145] Table 15: Optical density (OD) of secondary seed culture after shake flask incubation 600 The results included measurements of the volume required for resuspending the cell pellet. The master culture prepared in this manner was transferred to a Kuhner incubation shaker (Table 14) and incubated. Samples were taken from the shaker during incubation. Samples were taken under aseptic conditions to determine glucose, acetate, ammonium, anthranilic acid, and OD. 600 And dry biomass.

[0146] result Acetate concentration Table 16: An overview of acetate concentrations measured over time in two replicates for each of media 1 through 3. Samples using media 3 were not measured because no acetate was added. (nd = not measured; DL = limit of detection) ammonium concentration Table 17: Ammonium concentration (NH4+) over time in two replicates of each of media 1 to 3. + An overview of the concentration. D-glucose concentration Table 18: An overview of D-glucose concentrations measured over time in two replicates for each of media 1–3. The limit of detection (DL) is 0.3 mmol / L D-glucose. Dry biomass concentration (DBM) Table 19: Final dry biomass concentration in two replicates of each of media 1 to 3. o-aminobenzoic acid concentration (oAB) Table 20: An overview of the concentration of anthranilic acid over time in two replicates of each of media 1–3. The limit of detection (DL) is 0.2 g / L. Product yield Table 21: An overview of the yields of anthranilic acid at 30 h and 52 h (based on glucose) in two replicates of media 1 to 3. They are designated as quotients based on mass (g). oAB / g C-葡萄糖 ) and mole-based quotient (mol) oAB / mol 葡‍萄‍糖 ). Summarize In the study of the culture of anthranilic acid-producing bacteria based on Corynebacterium glutamicum in shake flasks, the effect of different concentrations of acetate (potassium acetate) as an additional carbon source on the yield of anthranilic acid (based on the amount of glucose used) was investigated.

[0147] It has been found that the final oAB titer and therefore the oAB yield (g) oAB / g 葡萄糖 (Based on the amount of glucose used) increases with increasing acetate concentration. Regarding the tested media, the highest yield was achieved with medium 2, which contains 20 g / L glucose and 13.299 g / L potassium acetate. Here, 0.186 g was achieved. oAB / g 葡萄糖 The value was 0.109 g in the absence of acetate (medium culture medium 3), while it only reached 0.109 g. oAB / g 葡萄糖 The value of .

Claims

1. A method for producing aminobenzoic acid, the method comprising the following steps: (A) Fermenting fermentable carbonaceous and nitrogenous compounds in the presence of microorganisms to obtain a fermentation broth containing aminobenzoate anions and / or aminobenzoic acid; (B) Obtaining 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; as well as (D) Add the reduction product from step (C) to the fermentation in step (A).

2. The method of claim 1, wherein the carbon dioxide formed during fermentation in step (A) is not converted into the reduction product in step (C).

3. The method of claim 1 or 2, wherein anthranilic acid or para-aminobenzoic acid is produced.

4. The method according to any one of claims 1 to 3, The method includes step (E), in which aminobenzoic acid from step (B) is decarboxylated to aniline, and the resulting carbon dioxide is converted into the reduction product in step (C).

5. The method of any one of claims 1 to 4, wherein carbon dioxide from an external source is converted into the reduction product in step (C).

6. The method of claim 5, wherein the carbon dioxide originates from an external source: • Processes used to produce (i) bioethanol, (ii) cement, (iii) hydrogen, or (iv) epoxides, or • Biodegradation processes in the digestive tract, or • The combustion of fuel, or • Used in the process of extracting carbon dioxide from the air.

7. The method of any one of claims 1 to 6, wherein step (C) comprises the reaction of carbon dioxide with hydrogen or the electrolysis of carbon dioxide.

8. The method according to any one of claims 1 to 7, The microorganisms in step (A) are selected from Escherichia coli (E. coli). Escherichia coli ), Pseudomonas putida ( Pseudomonas putida ), Corynebacterium glutamicum ( Corynebacterium glutamicum Bacillus coagulans ( Bacillus coagulans ), Cotton Asylum ( Ashbya gossypii Pichia pastoris ( Pichia pastoris ), Hansenula polymorpha ( Hansenula polymorpha ), Max Kluyveromycin ( Kluyveromyces marxianus ), Yarrowia lipolytica ( Yarrowia lipolytica Bayer conjugated yeast ( Zygosaccharomyces bailii ), brewer's yeast ( Saccharomyces cerevisiae ), or a mixture of two or more of the above-mentioned microorganisms, and / or The fermentable carbon-containing compounds are selected from starch hydrolysate, formic acid, alkali metal salts of formic acid, ammonium formate, acetic acid, alkali metal salts of acetic acid, ammonium acetate, sugarcane juice, beet juice, hydrolysate of lignocellulose raw materials, or a mixture of two or more of the above carbon-containing compounds. and / or The nitrogen-containing compounds are selected from ammonia, ammonia water, ammonium salts, soybean protein, urea, or a mixture of two or more of the above nitrogen-containing compounds.

9. The method of any one of claims 1 to 8, wherein the microorganism in step (A) is selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans, or a mixture of two or more of the above microorganisms.

10. The method of 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. The method of 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. The method of any one of claims 9 to 11, wherein the fermentable carbon-containing compound is selected from starch hydrolysate, alkali metal salt of formic acid, ammonium formate, alkali metal salt of acetic acid, ammonium acetate, sugarcane juice, beet juice, hydrolysate of lignocellulose raw material, or a mixture of two or more of the above carbon-containing compounds.

13. The method of any one of claims 1 to 8, wherein the microorganism in step (A) is selected from *Ashurus hygroscopicus*, *Pichia pastoris*, *Hansenula polymorpha*, *Kluyveromyces marx*, *Yersinia lipolytica*, *Bayer zygosacchari*, *Saccharomyces cerevisiae*, or a mixture of two or more of the above microorganisms.

14. The method of claim 13, wherein the reduction product is selected from formic acid or acetic acid.

15. The method of claim 13 or 14, wherein step (A) is carried out in a pH range of 3.0 to <5.5 to obtain a fermentation broth containing aminobenzoic acid and / or aminobenzoate anions.

16. The method of any one of claims 13 to 15, wherein the fermentable carbonaceous compound is selected from starch hydrolysate, formic acid, acetic acid, sugarcane juice, beet juice, hydrolysate of lignocellulose raw material, or a mixture of two or more of the above carbonaceous compounds.

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