Glycation of aminobenzoic acid in order to improve efficiency of microbial processes

Abstract

The present invention relates to the discovery that glycated aminobenzoic acid (AB) is significantly less toxic for microbial cells than free AB. The invention describes fermentation media and processes that allow glycation of AB and the efficiency of microbial processes in which AB is contained or formed in the medium to be increased.

Description

[0001] Glycation of aminobenzoic acid to improve the efficiency of microbial processes

[0002] The present invention is based on the discovery that glycated aminobenzoic acid (AB) is significantly less toxic to microbial cells than free AB. Therefore, fermentation media and processes are described that enable glycation of AB, thus increasing the efficiency of microbial processes in which AB is present or produced in the medium.

[0003] The production of ortho-aminobenzoic acid (oAB) by microbial fermentations is well known in the art (e.g., Balderas-Hernandez et al. (2009) “Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli,” Microbial Cell Factories 8: 19, WO 2015 / 124687 and WO 2017 / 102853). The fermentative production of para-aminobenzoic acid (pAB) has also been described (Kubota et al. (2016) “Production of para-aminobenzoate by genetically engineered Corynebacterium glutamicum and non-biological formation of an / V-glucosyl byproduct,” Metab. Eng. 38: 322-330). In this study, it was also observed that pAB was glycated in the presence of glucose. Since the study aimed at an efficient process for the fermentative production of pAB, the formation of this compound was considered undesirable.

[0004] Previously published studies have shown that pAB and oAB, as fermentation products, can be toxic to the producing microorganisms even at concentrations that must be achieved in the fermentation medium within the framework of an economically viable production process (WO 2015 / 124687 and Kubota, 2016). Thus, the economic viability of such AB production is limited by the achievable product concentrations—and thus the achievable space-time yield. Therefore, it is desirable to find ways to allow the presence of higher AB concentrations in a fermentation medium without impairing the metabolic activity of the AB-producing microorganisms. The state of the art proposes certain compositions of the fermentation media used to reduce the toxicity of oAB and pAB.The absence of ammonium ions (WO 2019 / 073035) or the presence of alkali ions (WO 2019 / 073033) can reduce the sensitivity of microorganisms to oAB and pAB. Nevertheless, there is a need for further methods to protect microorganisms from the effects of high AB concentrations. Averesch & Rothschild (2016, "Metabolic Engineering of Bacillus subtilis for production of para-aminobenzoic acid - unexpected importance of carbon source is an advantage for space application", Microbial Biotechnology, 12: 703-714) describe, without providing further details on concentrations and boundary conditions, that glycation of pAB can mitigate their toxicity. The study underlying the present application surprisingly found that glycation of AB can significantly reduce their toxicity. Glycation of AB in aqueous solution occurs spontaneously when reducing sugars are present.

[0005] Therefore, in a first embodiment, the present invention relates to an aqueous composition containing a total amount of aminobenzoic acid (AB) of at least 40 g / l, wherein at least 2 g / l of this total amount is present as glycated AB (gly-AB).

[0006] Preferably at least 3 g / l, more preferably at least 4 g / l, even more preferably at least 6 g / l and most preferably at least 8 g / l of the total amount of AB is present as glycated AB.

[0007] In a preferred embodiment of the present invention, at least 7.5%, more preferably at least 15%, and most preferably at least 20% of the total amount of AB is present as glycated AB.

[0008] Aqueous composition

[0009] The term "aqueous composition" refers to a mixture containing the above-mentioned components with water as a solvent. The presence of organic solvents is preferably limited to a maximum of 5 wt.%, more preferably a maximum of 2 wt.%, even more preferably a maximum of 1 wt.%, and most preferably a maximum of 0.5 wt.%, in each case based on the total amount of all solvents. Conversely, this means that the water content, based on the total amount of all solvents, is at least 95 wt.%, more preferably at least 98 wt.%, even more preferably at least 99 wt.%, and most preferably at least 99.5 wt.%.

[0010] The aqueous composition is preferably a nutrient medium that enables metabolic activity of the prokaryotic cells optionally present therein. Therefore, in a preferred embodiment of the present invention, the aqueous composition contains sources of inorganic nutrients usable by the prokaryotic cells used, in particular nitrogen, sulfur, and phosphorus, as well as the trace elements they require. Depending on the type of prokaryotic cells, the addition of vitamins and / or complex media components such as peptone or yeast extract may be useful. These complex media components can also simultaneously serve as nitrogen, sulfur, and / or phosphorus sources. The terms "nitrogen source," "sulfur source," and "phosphorus source" therefore do not necessarily refer to separate chemical compounds, but can also characterize different functions of a compound.Furthermore, the aqueous composition preferably contains at least one fermentable substrate that the prokaryotic cells in question can utilize as an energy and carbon source. In a particularly preferred embodiment of the present invention, the fermentable substrate is one of the reducing sugars defined further below in this application, in particular a sugar selected from the group consisting of glucose, fructose, mannose, galactose, maltose, lactose, and sucrose.

[0011] Particularly suitable culture media are described in the exemplary embodiments contained in this application. However, the skilled person is generally able to select suitable culture media based on the known physiological requirements of a microorganism.

[0012] Aminobenzoic acid

[0013] The term "aminobenzoic acid" (AB) is known to those skilled in the art. It refers to a derivative of benzoic acid characterized by the presence of an amino group bonded to the phenyl ring. The term aminobenzoic acid encompasses the two known structural isomers, which differ in the position of the amino group relative to the carboxyl group: ortho-aminobenzoic acid (oAB) and para-aminobenzoic acid (pAB). If one of the two isomers is specifically meant in this application, it is referred to using one of the aforementioned terms. The aforementioned compounds are molecules that can be positively charged by protonation of the amino group, negatively charged by deprotonation of the amino group, and have a neutral overall charge at the isoelectric point. Unless otherwise indicated in individual cases, the terms AB, oAB, and pAB refer to molecules of all charge states.

[0014] In this application, the terms "free AB", "free oAB" and "free pAB" are used to indicate that they are present without chemical modification, in particular not glycated. Conversely, the terms "total AB", "total oAB" and "total pAB" each refer to the sum of the molecules present with the corresponding basic structure, regardless of whether they are chemically modified by glycation or not.

[0015] Glycated AB

[0016] The term "glycation" refers to the covalent bonding of a sugar to the amino group of AB. This occurs preferably by reacting the aldehyde function of a reducing sugar with the amino group, thereby splitting off water. This reaction can be enzyme-catalyzed and can occur purely chemically without the involvement of enzymes. The reaction is reversible in aqueous solution and pH-dependent, so that the resulting glycated AB, oAB, or pAB can be split back into its starting materials by hydrolysis. Glycated AB then reconverts to free AB and the sugar bound during glycation.

[0017] Reducing sugar

[0018] In a preferred embodiment of the present invention, the aqueous composition contains at least one reducing sugar. The molar ratio of the total amount of aldehyde groups of the reducing sugar to total AB is at least 0.3:1.0, preferably at least 0.4:1, and even more preferably at least 0.5:1.

[0019] Since the reducing sugar is bound to the AB through glycation, a distinction must be made between total sugar and free sugar when specifying the sugar quantities or concentrations. Total sugar, or the total amount, indicates the sum of the reducing sugar present freely in the aqueous composition and the reducing sugar bound to AB through glycation. However, when "free reducing sugar" is mentioned, this refers to the portion of sugar that is not bound to AB and is present freely in the composition.

[0020] The term "reducing sugar" refers to all sugars that possess an aldehyde function and are therefore capable of glycating free AB. According to the invention, both simple sugars and polyvalent sugars can be used, as long as they possess an aldehyde function. Preferred simple sugars are glucose, fructose, xylose, arabinose, mannose, and galactose. Preferred polyvalent sugars are maltose, lactose, sucrose, and cellobiose. Particularly preferred reducing sugars are glucose, xylose, arabinose, and cellobiose. Glucose is most preferred. Reducing sugars that cannot be used by Corynebacterium as an energy or carbon source or to form a target product are xylose and cellobiose.

[0021] Prokaryotic cells

[0022] The aqueous composition contains prokaryotic cells.

[0023] In one embodiment, the aqueous composition contains a total amount of aminobenzoic acid (AB) of at least 40 g / l and several cells of the genus Corynebacterium.

[0024] In an alternative embodiment, the aqueous composition contains a total amount of aminobenzoic acid (AB) of at least 5 g / l and several cells of the genus Escherichia or Pseudomonas. The cell density of prokaryotic cells is preferably at least 10 4 Cells per ml. In a further preferred embodiment, the prokaryotic cells are present at a density of 2 g to 70 g dry mass per liter, more preferably between 10 g and 30 g dry mass per liter.

[0025] Regardless of the type of prokaryotic cells present in the aqueous composition, in a preferred embodiment of the present invention they are capable of converting a fermentable substrate to AB, particularly preferably to oAB.

[0026] Cells that possess this ability are preferably characterized by the physiological modification (i) defined below compared to the wild type. In a preferred embodiment of the present invention, the cell is additionally characterized by at least one of the modifications (iii), (iv) and (v). In a particularly preferred embodiment, a reduced activity of phosphoenolpyruvate carboxylase as defined in (ii) additionally characterizes the cell. These cells can be any prokaryotic cells suitable according to the invention, since the shikimate pathway used here is phylogenetically highly conserved. However, they are preferably cells of the genus Corynebacterium, preferably Corynebacterium glutamicum and particularly preferably Corynebacterium glutamicum ATCC 13032.

[0027] (i) An activity of the anthranilate phosphoribosyltransferase that is reduced compared to the respective wild type, although residual activity must be present. The residual activity is preferably between 10% and 60%, more preferably between 20% and 50% of the activity natively present in the respective wild type. This is preferably achieved by a reduced expression of the gene for anthranilate phosphoribosyltransferase (trpD) compared to the wild type, although expression is not completely suppressed. This is preferably achieved by using a promoter sequence that has lower transcriptional activity than the endogenously present promoter sequence, or by modifying the distance between the ribosome binding site and the start codon of the trpD gene, or by altering 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 having an altered ribosomal binding site and optionally an altered 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) Deletion or inactivation of the gene encoding the phosphoenolpyruvate carboxylase as defined in SEQ ID NO. 4.This can be done in any way familiar to the skilled person, preferably by deleting the gene or a partial sequence thereof, by introducing at least one stop codon, or by deleting or inactivating the promoter sequence. Particular preference is given to deleting at least part of the protein-coding sequence of the gene (SEQ. ID NO. 5).

[0028] (iii) 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 any method known to the person skilled in the art, in particular by introducing multiple copies of the corresponding gene into the microorganism or by using stronger promoters to express the endogenously present enzyme. A particularly preferred promoter for the expression of foreign genes or the increased expression of endogenous genes is ptuf as defined in SEQ ID NO. 8.

[0029] (iv) 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.

[0030] (v) presence of a 3-deoxyarabinoheptulosanate 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.

[0031] In a preferred embodiment of the present invention, the prokaryotic cell defined above additionally exhibits increased activity of xylose isomerase and xylulokinase. This increased activity preferably occurs through enhanced expression of the two genes. This enhanced expression can be achieved by any method known to the person skilled in the art, in particular by introducing multiple copies of the corresponding gene into the microorganism or by using stronger promoters to express the endogenously present enzyme. According to the invention, increased expression of enzymes with amino acid sequences as defined in SEQ ID NO. 12 or SEQ ID NO. 14, or variants thereof, is preferred.

[0032] With reference to anthranilate phosphoribosyltransferase (SEQ ID NO. 3), shikimate kinase (SEQ ID NO. 6 or 7), 3-phosphoshikimate-l-carboxyvinyltransferase (SEQ ID NO. 9), chorismate synthase (SEQ ID NO. 10), xylose isomerase (SEQ ID NO. 12), and xylulokinase (SEQ ID NO. 14), "variant" means an enzyme obtained by adding, deleting, or exchanging up to 10%, preferably up to 5%, of the amino acids contained in the respective enzyme. In principle, the aforementioned modifications can occur continuously or discontinuously at any desired position in the enzyme. However, they preferably occur only at the N-terminus and / or C-terminus of the polypeptide. Substitutions of amino acids are preferably conservative substitutions, i.e. those in which the changed amino acid has a residue with similar chemical properties to the amino acid present in the unchanged enzyme.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 is preferably at least 80% of the specific activity of the unaltered enzyme. Enzyme tests for detecting the activity of the aforementioned enzymes can be found in the literature by those skilled in the art.

[0033] With reference to DAHP synthase (SEQ ID NO. 11), "variant" means an enzyme obtained by adding, deleting, or exchanging up to 5%, preferably up to 2%, of the amino acids contained in the respective enzyme, with the proviso that positions 76 and 211 remain unchanged. It is further preferred that positions 10, 13, 147, 148, 150, 151, 179, 209, 211, and 212 also remain unchanged. In a more preferred embodiment of the present invention, positions 144, 175, and 215 are also unchanged in the variant. In an even more preferred embodiment of the present invention, in addition to the aforementioned positions, positions 92, 97, 165, 186, and 268 are also unchanged. The person skilled in the art understands that the aforementioned positions shift accordingly when amino acids are deleted or inserted.In principle, the aforementioned modifications can occur continuously or discontinuously at any desired position in the enzyme. However, they preferably occur only at the N-terminus and / or the C-terminus of the polypeptide. Substitutions of amino acids are preferably conservative substitutions, i.e. those in which the modified amino acid has a residue with similar chemical properties to the amino acid present in the unchanged enzyme. Thus, particular preference is given to exchanging amino acids with basic residues 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 non-polar residues for those with non-polar residues. It is particularly preferred that the variant of DAHP synthase also possesses corresponding enzyme activity.Particularly preferably, the specific enzyme activity of the variant is at least 80% of the specific activity of the unmodified DAHP synthase according to SEQ. ID NO. 11.

[0034] Metabolic activity

[0035] In a preferred embodiment of the present invention, the prokaryotic cells present in the aqueous composition are metabolically active. The term "metabolic activity" refers to a state of the prokaryotic cells in which they take up a fermentable substrate and biochemically convert it to maintain their vital functions.

[0036] In a preferred embodiment of the present invention, metabolic activity is defined by growth, either as an increase in the number of cells or as an increase in the dry biomass of the aqueous composition. At a constant volume of the aqueous composition, this means an increase in the cell density or the dry biomass per unit volume. With increasing volume, in particular through the addition of fresh substrate solution, or with partial replacement of the aqueous composition, these changes must be taken into account when calculating the number of cells or the dry biomass. The number of cells or the dry biomass are preferably determined via the change in the light absorption of the culture medium at a wavelength of 600 nm or by determining the backscattering. The latter is preferably carried out at a wavelength of 620 nm. The conversion factor between the dry biomass or cell density and absorption can be determined using methods familiar to the person skilled in the art.In a more preferred embodiment, the metabolic activity is defined by the consumption of the at least one fermentable substrate in the aqueous composition.

[0037] The study underlying this application has shown that the growth rate of prokaryotic cells is at least 30% higher in the presence of a defined concentration of glycated AB than in the presence of the same concentration of the sodium salt of free AB. Regarding the effect of the aqueous composition according to the invention, it should be noted that the AB contained therein will generally not be fully glycated, so the effect may be lower in the actual application of the composition according to the invention.

[0038] In another preferred embodiment of the present invention, the metabolic activity is defined as the production of aminobenzoic acid. Toxicity of AB

[0039] The term "AB toxicity" refers to the adverse effect that aminobenzoic acid has on the metabolic activity of prokaryotic cells above a certain concentration. Above a certain threshold, which depends on the organism and the media components used (see WO 2019 / 073035 and WO 2019 / 073033), the activity of the prokaryotic cells is increasingly inhibited by increasing AB concentrations. In extreme cases, this leads to cell death.

[0040] In the study underlying this patent application, it was surprisingly found that the specific toxicity of glycated AB is lower than the specific toxicity of free AB, i.e., a given molar concentration of free AB in the nutrient medium inhibits the metabolic activity of a prokaryotic cell more strongly than the same molar concentration of glycated AB. Therefore, it has proven advantageous to create conditions in a nutrient medium that promote glycation of free AB. Due to the influence of other media components on AB toxicity, this comparison is preferably carried out using media that differ only in the concentrations of free and glycated AB and, in particular, otherwise have identical concentrations of sodium and ammonium ions.

[0041] Use of the aqueous composition

[0042] In a further embodiment, the present application relates to the use of the above-defined aqueous composition for culturing prokaryotic cells.

[0043] All definitions given above for the aqueous composition also apply to this embodiment, unless explicitly stated otherwise.

[0044] The term "cultivation," when applied to prokaryotic cells, refers to incubation in an aqueous composition, particularly in a nutrient medium suitable for the cells in question, under conditions that allow metabolic activity of the cells. This requires, in particular, that the aqueous composition contains all the components required by the cells, that the pH is maintained within a range acceptable to the respective cells, and that the temperature is maintained within an acceptable range, preferably between 20°C and 40°C.

[0045] Use of reducing sugars

[0046] In yet another embodiment, the present invention relates to the use of reducing sugars in concentrations of at least 10 g / l, preferably at least 20 g / l, and more preferably at least 30 g / l total sugars to reduce the toxicity of AB. Method for reducing the toxicity of AB

[0047] In yet another embodiment, the present invention relates to a method for reducing the toxicity of AB, comprising the step of contacting free AB in aqueous solution with a reducing sugar. The molar ratio of the total amount of aldehyde groups of the reducing sugar to total AB is at least 0.3:1.0, preferably at least 0.4:1, and even more preferably at least 0.5:1.

[0048] All definitions given above also apply to this embodiment. The definitions for the aqueous composition also apply to the aqueous solution mentioned here.

[0049] Contacting can be carried out in any manner known to those skilled in the art. In a preferred embodiment of the present invention, the sugar is dissolved in the aqueous solution, particularly preferably in a nutrient medium. The nutrient medium preferably contains prokaryotic cells that convert a fermentable substrate contained in the nutrient medium into AB. The AB formed by the prokaryotic cells is secreted by the cells and then comes into direct contact with the reducing sugar already present in solution.

[0050] Method for culturing a prokaryotic cell

[0051] In a further preferred embodiment, the present invention relates to a method for cultivating prokaryotic cells in the presence of AB and at least one reducing sugar, wherein a) the cells are those of the genus Corynebacterium and the total amount of aminobenzoic acid (AB) is at least 40 g / l; or b) the cells are those of the genus Escherichia or Pseudomonas and the total amount of aminobenzoic acid (AB) is at least 5 g / l; and the molar ratio of the total amount of aldehyde groups of the reducing sugar to total AB is at least 0.3:1.0.

[0052] All definitions given above also apply to this embodiment.

[0053] The prokaryotic cell is preferably a cell capable of converting a fermentable substrate into AB. It is particularly preferably capable of using the reducing sugar used as a fermentable substrate for this purpose. Therefore, the preferred cultivation method is one in which the prokaryotic cells form AB, i.e., a production method for AB through microbial fermentation.

[0054] The incubation of microbial cells in a medium in which a portion of the AB present is glycated can reduce the toxicity of the AB. This is relevant for all microbial processes in which AB is present in the medium. This primarily applies to processes for the production of oAB or pAB by microbial fermentation. In such processes, AB accumulates as intended, and it is advantageous for the efficiency of such processes if the fermentation can be carried out for as long as possible, i.e. until the highest possible AB concentrations are reached, without impairing the activity of the cells. The reduction of the toxicity of AB through glycation is also active for processes in which no AB is produced but the microorganisms still come into contact with it.These are particularly processes in which the AB is not the desired product, but rather the starting material to be converted into a target product through microbial fermentation. Overall, the present invention offers advantages whenever microbial cells come into contact with AB and a reduction in their metabolic activity or viability is undesirable.

[0055] The following embodiments serve only to illustrate the present invention. They are not intended to limit the scope of the patent claims in any way.

[0056] Examples of implementation

[0057] Example 1: Reaction of glucose and oAB to gly-oAB

[0058] By neutralization with 5 M NaOH, 150 g / L of oAB was dissolved in water and adjusted to pH 7.0. Separately, 120 g / L of glucose was dissolved in water and also adjusted to pH 7.0. A total of nine different batches were prepared from various proportions of these two solutions and water, with glucose concentrations of 10, 30, and 60 g / L, respectively, and oAB concentrations of 5, 40, and 75 g / L, respectively. These batches were prepared in duplicate in 2 mL reaction tubes and incubated in a Thermo Mixer® C (Eppendorf) at 1000 rpm and 33 °C. The course of the reaction between glucose and oAB was monitored by determining the remaining free glucose using a Cedex Bio Analyzer (Roche). A decrease in the measured glucose concentration in mol / L corresponds to an increase in the gly-oAB concentration in mol / L. After approximately 75 hours, no further change in glucose concentration was observed.

[0059] Table 1 shows the proportion of gly-oAB in the total amount of oAB used after 75 h for the different batches. Depending on the initial glucose and oAB concentration, approximately 8.8–87.3% of the total oAB present is present as glycated molecules at equilibrium.

[0060] Table 1: Proportion of gly-oAB in the total amount of oAB used after 75 h for different starting concentrations of glucose and oAB.

[0061] Example 2: Reaction of glucose and pAB to gly-pAB

[0062] By neutralization with 5 M NaOH, 150 g / L of pAB was dissolved in water and adjusted to pH 7.0. Separately, 120 g / L of glucose was dissolved in water and also adjusted to pH 7.0. A total of nine different preparations were prepared from various proportions of these two solutions and water, with glucose concentrations of 10, 30, and 60 g / L, respectively, and pAB concentrations of 5, 40, and 75 g / L, respectively. These preparations were prepared in duplicate in 2 mL reaction tubes and incubated in a ThermoMixer® C (Eppendorf) at 1000 rpm and 33 °C. The course of the reaction between glucose and pAB was monitored by determining the remaining free glucose using a Cedex Bio Analyzer (Roche). A decrease in the measured glucose concentration in mol / L corresponds to an increase in the gly-pAB concentration in mol / L. After approximately 75 hours, no further change in glucose concentration was observed.

[0063] Table 2 shows the proportion of gly-pAB in the total amount of pAB used after 75 h for the different batches. Depending on the initial glucose and pAB concentration, approximately 7.8–83.3% of the total pAB present is present as glycated molecules at equilibrium.

[0064] Table 2: Proportion of gly-pAB in the total amount of pAB used after 75 h for different starting concentrations of glucose and pAB.

[0065] Example 3: Determination of gly-oAB and gly-pAB by acid reverse reaction

[0066] Due to the metabolism of glucose in fermentation processes, glycated aminobenzoic acid cannot be quantified via a decrease in glucose concentration, as this decrease may also be due to glucose consumption. However, under acidic conditions, a reverse reaction of gly-oAB or gly-pAB to free glucose and aminobenzoic acid is possible. This allows quantification of gly-oAB or gly-pAB via the free glucose concentration present before and after the reverse reaction, provided the reverse reaction proceeds almost completely.

[0067] To test this, various glucose-aminobenzoic acid mixtures were incubated for approximately 75 hours, as described in Examples 1 and 2, to form glycated aminobenzoic acid. 200 pL of the respective mixture was then mixed with 800 pL of 1 M hydrochloric acid and allowed to stand at room temperature. Free glucose was measured at regular intervals using a Cedex Bio Analyzer (Roche). After approximately 2 hours, no further change in the glucose concentration was detectable.

[0068] Table 3 shows the percentage of free glucose measured after acidic reverse reaction relative to the initial glucose concentration used for the reverse reaction of gly-pAB. Regardless of the glucose and pAB concentrations used, more than 95.4% of the glucose present can be recovered after acid treatment. Table 3: Reverse reaction of gly-pAB under acidic conditions

[0069] Table 4 shows the percentage of free glucose measured after acid back-reaction relative to the initial glucose concentration used for the back-reaction of gly-oAB. As the oAB concentration increases or decreases, the proportion of glucose present that can be recovered after acid treatment decreases. However, at glucose concentrations of 30 g / L or more and oAB concentrations up to 75 g / L, more than 88.2% of the glucose present can be recovered. The acid back-reaction can therefore be used to quantify gly-pAB and, with limitations, gly-oAB.

[0070] Table 4: Reverse reaction of gly-oAB under acidic conditions

[0071] Example 4: Demonstration of reduced toxicity of glycated oAB or pAB to microorganisms

[0072] General information on experimental design

[0073] For the following examples, the BioLector Pro micro-cultivation system from m2p-labs (Aachen, Germany) was used, which allows 48 parallel cultivations in one microtiter plate. The bacterial strains were cultured in an initial volume of 800 pL and a final volume of 1500 pL of medium in 48-well BOH2 flower plates (m2p-labs, Aachen, Germany) at 1000 rpm (shaking angle 3 mm). The plates used contain immobilized indicator substances (optodes), which enable online measurement of the pH value and the oxygen partial pressure (pO2) in each well. Growth was monitored for all cultivations at 620 nm by measuring the scattered light signal (gain set to 3). The parallel cultivation system from m2p-labs is well-known and suitable for the cultivation of bacterial strains.

[0074] The cultivation temperature and media used depend on the bacterial strain being investigated and are listed in Table 5 along with other cultivation parameters. For the examples, the bacterial wild-type strains Corynebacterium glutamicum ATCC 13032, Escherichia coli K12, and Pseudomonas putida DSM 6125 were selected. For C. glutamicum, CG XII minimal medium is used in the main cultivation, while for E. coli and P. putida, LB complex medium (Miller's LB broth (L3522, Sigma-Aldrich, St. Louis, USA)) is used, which is typically used for the cultivation of this and related species.

[0075] The exact media compositions with adjustments are described in more detail in Table 6.

[0076] Table 5: Pre-culture management, media used and cultivation parameters of the examples shown below.

[0077] Table 6: Media composition and stock solutions

[0078] To determine the toxic effects of free oAB or free pAB compared to the glycated form gly-oAB or gly-pAB, respectively, after an initial growth phase, additional main culture media with different concentrations of oAB and gly-oAB or pAB and gly-pAB were added (hereinafter referred to as spikes). The preparation of main culture media with the gly-oAB or gly-pAB form was based on the kinetics in Examples 1 and 2 and was carried out for 70 h with stirring. Media without AB were added as a reference (hereinafter referred to as reference spikes).

[0079] Since the gly-oAB or gly-pAB sp / kes leads to an increase in the amount of carbon added in the form of glucose bound in the complex and still free from the equilibrium reaction, the same amount of glucose was also added to the reference sp / kes as well as the oAB and pAB spikes, but without prior incubation, so that the complex between oAB or pAB and glucose had not yet formed at the time of addition. This was verified experimentally using the method shown in Example 3.

[0080] Tables 7 (oAB) and 8 (pAB) provide an overview of the experimental approaches. For each approach, the type and amount of the different solutions added after the growth phase are listed. The amount refers to a single culture well of the microtiter plate. While 800 pL of inoculated medium was used at the beginning of the cultivation, a total of 700 pL of solutions were added to each well after the growth phase. Additionally, the resulting AB concentration after spiking and the increase in glucose concentration in the well due to the spike are given. The composition and preparation of the solutions used are also described in Table 6. Table 7: Overview of the experimental approaches for investigating the toxicity of oAB and gly-oAB

[0081] Table 8: Overview of experimental approaches to investigate the toxicity of pAB or gly-pAB

[0082] Example 4.1: Toxicity of gly-oAB versus free oAB for Corynebacterium glutamicum ATCC 13032

[0083] For the following example, the strain Corynebacterium glutamicum ATCC 13032 was cultured in 800 pL of minimal medium CG XII in the m2p-labs BioLector Pro microculture system as described above. After 7.2 h, different concentrations of oAB and gly-oAB were added. All assays and references were performed in triplicate (Table 7: Example 4.1).

[0084] The toxicity of oAB or gly-oAB was investigated based on growth compared to two controls without oAB / gly-oAB-Sp / ke (Reference 1 and Reference 2). For this purpose, the slope of the scattered light signal was analyzed in the first 10 hours after the spike (Table 9). Both oAB and gly-oAB lead to a delayed growth. However, at the same concentration of oAB in the oAB or gly-oAB spike, a lower toxicity of gly-oAB compared to free oAB was evident at all concentrations, based on a higher slope in the scattered light signal.

[0085] Table 9: Slope of the scattered light signal of a cultivation of the strain C. glutamicum ATCC 13032 in the first 10 h after spiking with oAB, gly-oAB or a mixture of medium and glucose solution (reference).

[0086] Example 4.2: Toxicity of gly-oAB compared to free oAB for Escherichia coli K12 and Pseudomonas putida DSM 6125

[0087] For the following example, the strains Escherichia coli K12 and Pseudomonas putida DSM 6125 were cultivated in 800 pL LB medium in the BioLector Pro micro-cultivation system from m2p-labs as described above. After 4 h (E. coli) and 7 h (P. putida), different concentrations of oAB and gly-oAB were added, respectively. All assays and references were performed in triplicate (Table 7: Example 4.2). Tables 10 and 11 show the slope of the scattered light signal in the first 2 h after the spike for E. coli and in the first 10 h for P. putida. In both organisms, both oAB and gly-oAB have a growth-inhibiting / toxic effect compared to the references. In E. coli, at the lowest oAB concentration (5 g / L), the growth losses of oAB and gly-oAB are similar. However, at 10 and 15 g / L oAB, the lower toxicity of gly-oAB compared to free oAB is evident, as evidenced by the higher slope of the scattered light signal. In P.putida, the slope of the scattered light signal indicates that gly-oAB is less toxic than free oAB, even at the lowest concentration (5 g / L). At 10 g / L of free oAB, no growth occurs after the spike, whereas cells continue to grow at the same concentration of gly-oAB. At the highest concentration (15 g / L), neither free oAB nor gly-oAB exhibits any growth after the spike.

[0088] The reduction of the toxic effect by glycation of oAB can therefore be demonstrated not only for C. glutamicum ATCC 13032, but also for E. coli K12 and P. putida DSM 6125.

[0089] Table 10: Slope of the scattered light signal of a cultivation of the Escherichia coli K12 strain in the first 2 h after spiking with oAB, gly-oAB or a mixture of medium and glucose solution (reference).

[0090] Table 11: Slope of the scattered light signal of a cultivation of the strain Pseudomonas putida DSM 6125 in the first 10 h after spiking with oAB, gly-oAB or a mixture of medium and glucose solution (reference). 15 g / L oAB (gly-oAB) -0.12

[0091] Example 4.3: Toxicity of gly-pAB versus free pAB for Corynebacterium glutamicum ATCC 13032, Escherichia coli K12 and Pseudomonas putida DSM 6125

[0092] The toxicity of pAB and gly-pAB was investigated as previously described in Examples 4.1 and 4.2 for oAB and gly-oAB, respectively, and as shown in Table 8. The results are summarized in Tables 12, 13, and 14. The slope of the scattered light signal after the spike is shown for all three strains.

[0093] In all cases, growth losses were observed when free pAB or gly-pAB were added compared to the reference cultures without pAB. For C. glutamicum ATCC 13032, the reduced toxicity of gly-pAB compared to free pAB was demonstrated under all conditions by the slope of the scattered light signal (Table 12), analogous to previous data for gly-oAB and oAB (Table 9).

[0094] Table 12: Slope of the scattered light signal of a culture of the strain Corynebacterium glutamicum ATCC 13032 in the first 4.7 h after spiking with pAB, gly-pAB or a mixture of medium and glucose solution (reference).

[0095] For E. coli K12, no advantage of gly-pAB over free pAB was observed after 4 h of addition of 5 g / L pAB. Since the glucose in pAB-Sp / ke is unbound and thus accessible to the microorganism and usable as a carbon source, a higher gradient is observed for pAB-Sp / ke compared to gly-pAB-Sp / ke. At 10 and 15 g / L, the lower toxic effect of the glycated form is evident from the higher gradient of the scattered light signal compared to pAB-Sp / ke. Table 13: Slope of the scattered light signal of a culture of the Escherichia coli K12 strain in the first 4 h after spiking with pAB, gly-pAB, or a mixture of medium and glucose solution (reference). For P. putida DSM 6125, no difference between pAB and gly-pAB was observed after 5 h of addition of 5 and 10 g / L pAB. However, at 15 g / L, the lower toxic effect of the glycated form is evident from the higher slope of the scattered light signal compared to the pAB-sp / ke.

[0096] Table 14: Slope of the scattered light signal of a culture of the strain Pseudomonas putida DSM 6125 in the first 8 h after spiking with pAB, gly-pAB or a mixture of medium and glucose solution (reference).

Claims

Patent claims 1. An aqueous composition comprising a) a total amount of ortho-aminobenzoic acid (oAB) of at least 40 g / l and several cells of the genus Corynebacterium; or b) a total amount of ortho-aminobenzoic acid (oAB) of at least 5 g / l and several cells of the genus Escherichia or Pseudomonas; wherein at least 2 g / l of this total amount is present as glycated AB (gly-AB).

2. The aqueous composition of claim 1, wherein at least 15% of the total amount of oAB is present as glycated oAB.

3. The aqueous composition according to claim 1 or 2, additionally containing at least one reducing sugar, wherein the molar ratio of the total amount of aldehyde groups of the reducing sugar to total oAB is at least 0.3:1.

0.

4. The aqueous composition according to claim 3, containing at least one reducing sugar which cannot be utilized by the cells contained in the aqueous composition as an energy or carbon source or for the formation of a target product.

5. The aqueous composition according to any one of claims 1 to 4, additionally containing a nitrogen source usable by the cells, a sulfur source usable by the contained cells and a phosphorus source usable by the contained cells.

6. The aqueous composition according to any one of claims 1 to 5, wherein the cells are present in a concentration of 2 g to 70 g dry matter per liter.

7. The aqueous composition according to any one of claims 1 to 6, wherein the cells are metabolically active.

8. Use of the aqueous composition according to any one of claims 1 to 6 for cultivating the cells contained therein.

9. A process for reducing the toxicity of aminobenzoic acid (AB) comprising the step of contacting free AB in aqueous solution with a reducing sugar, wherein the molar ratio of the total amount of aldehyde groups of the reducing sugar to total AB is at least 0.3 : 1.

0.

10. The method according to claim 9, wherein the free AB to be detoxified in the aqueous solution is produced by the cells contained in the aqueous composition.

11. The use of reducing sugars in concentrations of at least 10 g / l total sugars to reduce the toxicity of AB.

12. The use according to claim 11, wherein the molar ratio of the total amount of aldehyde groups of reducing sugar to total AB is at least 0.3:1.

0.

13. A process for cultivating prokaryotic cells in the presence of ortho-aminobenzoic acid (oAB) and at least one reducing sugar, wherein a) the cells are those of the genus Corynebacterium and the total amount of aminobenzoic acid (AB) is at least 40 g / l; or b) the cells are those of the genus Escherichia or Pseudomonas and the total amount of ortho-aminobenzoic acid (oAB) is at least 5 g / l; and the molar ratio of the total amount of aldehyde groups of the reducing sugar to total oAB is at least 0.3:1.

0.

14. The method of claim 13, wherein the cells are capable of converting a fermentable substrate to oAB.

15. The method of claim 14, wherein the cells are capable of using the reducing sugar as a fermentable substrate.

16. The method according to claim 13 or 14, wherein at least one reducing sugar is present which cannot be utilized by the prokaryotic cells as an energy or carbon source.

Images