Saccharification of aminobenzoic acid for improved microbiological process efficiency

Abstract

The present invention relates to the discovery that saccharified aminobenzoic acid has a significantly lower toxicity to microbial cells than free aminobenzoic acid (AB). Fermentation media and methods are described that enable the saccharification of AB to increase the efficiency of microbial processes that contain or form AB in the media.

Description

[0001] The present invention is based on the discovery that saccharified aminobenzoic acid (AB) is significantly less toxic to microbial cells than free aminobenzoic acid (AB). Accordingly, the present invention describes fermentation media and methods that enable saccharification of AB, thereby increasing the efficiency of microbial processes in which AB is present or formed in the culture medium.

[0002] The preparation of anthranilate (oAB) by microbial fermentation is well known in the prior 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 fermentation preparation of p-aminobenzoic acid (pAB) has also been documented (Kubota et al. (2016) "Production of para-aminobenzoate by genetically engineered Corynebacterium glutamicum and non-biological formation of an N-glucosylbyproduct", Metab. Eng. 38:322-330). In this study, it was also observed that pAB undergoes saccharification in the presence of glucose. Since the focus of this study was on an efficient method for fermentative preparation of pAB, the formation of this compound was considered to be undesirable.

[0003] Previously published studies have shown that pAB and oAB, fermentation products, can become toxic to the producing microorganisms at the concentrations required for economically viable production processes in the fermentation medium (WO 2015 / 124687 and Kubota, 2016). Consequently, the economic viability of such AB production is limited by the achievable product concentrations—and, therefore, the achievable space-time yields. Therefore, it is desirable to develop methods that allow higher concentrations of AB to be present in the fermentation medium without impairing the metabolic activity of the AB-producing microorganisms. Prior art methods have proposed reducing the toxicity of oAB and pAB by using specific fermentation medium compositions. The absence of ammonium ions (WO 2019 / 073035) or the presence of alkali metal ions (WO 2019 / 073033) can reduce microbial sensitivity to oAB and pAB. However, other methods for protecting microorganisms from high AB concentrations remain desirable. 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) stated that glycation of pAB can reduce its toxicity, but did not specify further details regarding concentration and boundary conditions.

[0004] In the studies forming the basis of the present application, it has been found that, surprisingly, the saccharification of AB can significantly reduce its toxicity.Saccharification of AB occurs spontaneously in aqueous solution in the presence of reducing sugars.

[0005] Thus, in a first embodiment, the present invention relates to an aqueous composition comprising a total amount of aminobenzoic acid (AB) of at least 40 g / l, wherein at least 2 g / l of said total amount is in the form of 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 in the form of saccharified 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 in the form of saccharified AB.

[0008] Aqueous composition

[0009] The term "aqueous composition" refers to a mixture containing the above-mentioned components in water as a solvent. The presence of organic solvents is preferably limited to no more than 5% by weight, more preferably no more than 2% by weight, even more preferably no more than 1% by weight, and most preferably no more than 0.5% by weight, in each case based on the total amount of all solvents. This means, in turn, that the proportion of water, based on the total amount of all solvents, is at least 95% by weight, more preferably at least 98% by weight, even more preferably at least 99% by weight, and most preferably at least 99.5% by weight.

[0010] The aqueous composition is preferably a growth medium, which enables the prokaryotic cells optionally present therein to be metabolically active. Therefore, in a preferred embodiment of the present invention, the aqueous composition comprises a source of inorganic nutrients (particularly nitrogen, sulfur and phosphorus) that can be utilized by the prokaryotic cells used, and the trace elements required for the prokaryotic cells. Depending on the type of the prokaryotic cells, it may be useful to add vitamins and / or compound culture medium components (such as peptone or yeast extract). The compound culture medium components may also be used as nitrogen source, sulfur source and / or phosphorus source simultaneously. Therefore, the terms "nitrogen source", "sulfur source" and "phosphorus source" do not necessarily refer to independent compounds, but may also represent the different functions of the same compound.

[0011] Furthermore, the aqueous composition preferably comprises at least one fermentable substrate which the prokaryotic cells can use 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 herein below, in particular a sugar selected from the group consisting of glucose, fructose, mannose, galactose, maltose, lactose and sucrose.

[0012] Particularly suitable growth media are described in the working examples of the present application. However, a person skilled in the art is in principle able to select a suitable growth medium based on the known physiological requirements of the microorganisms.

[0013] aminobenzoic acid

[0014] The term "aminobenzoic acid" (AB) is known to those skilled in the art. It refers to a benzoic acid derivative, which is characterized by the presence of an amino group bound to the phenyl ring. The term "aminobenzoic acid" includes two known structural isomers, which differ in the position of the amino group relative to the carboxyl group: o-aminobenzoic acid (oAB) and p-aminobenzoic acid (pAB). If one of the two isomers is specifically mentioned in this application, it is represented by one of the above-mentioned terms. The above-mentioned compounds are molecules that can be in a positively charged state due to protonation of the amino group, in a negatively charged state due to deprotonation of the amino group, and in a state of overall neutral charge at the isoelectric point. Unless otherwise stated in individual cases, the terms "AB", "oAB" and "pAB" refer to molecules in all charge states.

[0015] In this application, the terms "free AB," "free oAB," and "free pAB" are used hereinafter to indicate that they have not been chemically modified, in particular not saccharified. In contrast, the terms "total amount of AB," "total amount of oAB," and "total amount of pAB" each refer to the sum of all molecules present having the corresponding basic structure, regardless of whether they have been chemically modified by saccharification.

[0016] Glycation AB

[0017] The term "saccharification" refers to the covalent binding of sugars to the amino groups of AB. This is preferably achieved by reacting the aldehyde functional groups of the reducing sugar with the amino groups, eliminating water. This reaction can be performed enzymatically or by purely chemical methods without enzymes. The reaction is reversible and pH-dependent in aqueous solution, meaning that the resulting saccharified AB, oAB, or pAB can be cleaved back into the starting materials by hydrolysis. The saccharified AB then regenerates free AB and the sugars bound during the saccharification process.

[0018] reducing sugars

[0019] In a preferred embodiment of the present invention, the aqueous composition comprises at least one reducing sugar. The molar ratio of the total amount of reducing sugar aldehyde groups to the total amount of AB is at least 0.3:1.0, preferably at least 0.4:1, and even more preferably at least 0.5:1.

[0020] Because reducing sugars bind to AB through saccharification, it is important to distinguish between total sugars and free sugars when specifying the amount or concentration of sugars. Total sugars or the total amount represents the sum of the free reducing sugars and the reducing sugars bound to AB through saccharification in the aqueous composition. In contrast, the term "free reducing sugars" refers to the portion of sugars in the composition that are free and not bound to AB.

[0021] The term "reducing sugar" refers to all sugars that have an aldehyde functional group and are therefore capable of saccharifying free AB. According to the present invention, both monosaccharides and oligosaccharides can be used as long as they have an aldehyde functional group. Preferred monosaccharides are glucose, fructose, xylose, arabinose, mannose and galactose. Preferred oligosaccharides are maltose, lactose, sucrose and cellobiose. Particularly preferred reducing sugars are glucose, xylose, arabinose and cellobiose. Glucose is very particularly preferred. Reducing sugars that cannot be used as an energy or carbon source or for forming the target product by Corynebacterium are xylose and cellobiose.

[0022] prokaryotic cells

[0023] The aqueous composition comprises prokaryotic cells.

[0024] In one embodiment, the aqueous composition comprises aminobenzoic acid (AB) in a total amount of at least 40 g / l and a plurality of cells of the genus Corynebacterium.

[0025] In an alternative embodiment, the aqueous composition comprises aminobenzoic acid (AB) in a total amount of at least 5 g / l and a plurality of cells of the genus Escherichia or Pseudomonas.

[0026] The cell density of the prokaryotic cells is preferably at least 10 4 In another preferred embodiment, the prokaryotic cells are present at a density of 2 g to 70 g dry matter per liter, more preferably 10 g to 30 g dry matter per liter.

[0027] In a preferred embodiment of the present invention, regardless of the type of prokaryotic cells present in the aqueous composition, they are capable of converting a fermentable substrate to form AB, particularly preferably oAB.

[0028] The cell of this ability is preferably characterized in that, compared with wild type, has physiological modification (i) defined below.In a preferred embodiment of the present invention, described cell characteristic also is to have modification (iii), (iv) and (v) at least one.In a particularly preferred embodiment, the activity reduction of phosphoenolpyruvate carboxylase as defined in (ii) is the other feature of described cell.Because the shikimic acid pathway utilized herein is highly conservative on phylogeny, therefore described cell can be according to all suitable prokaryotic cells of the present invention.But preferably Corynebacterium, preferably Corynebacterium glutamicum (Corynebacterium glutamicum), and the cell of Corynebacterium glutamicum ATCC13032 is particularly preferred.

[0029] (i) Compared with the corresponding wild type, the activity of anthranilate phosphoribosyltransferase was decreased

[0030] The expression of the anthranilate phosphoribosyltransferase (trpD) gene is preferably reduced compared to the wild type, but the expression is not completely suppressed. This is preferably achieved by:

[0031] Using a promoter sequence with lower transcriptional activity than the endogenous promoter sequence, or changing the distance from the ribosome binding site to the start codon of the trpD gene, or changing the start codon itself. In a preferred embodiment of the present invention, the activity of anthranilate phosphoribosyltransferase is reduced by deleting or inactivating the endogenous anthranilate phosphoribosyltransferase (trpD) gene and replacing it with a promoter sequence such as SEQ ID

[0032] The gene is replaced by an anthranilate phosphoribosyltransferase gene having a modified ribosome binding site and optionally a modified start codon as defined in SEQ ID NO. 1 or 2 (preferably SEQ ID NO. 2). The amino acid sequence of the anthranilate phosphoribosyltransferase preferably corresponds to endogenous anthranilate phosphoribosyltransferase, more preferably as defined in SEQ ID NO. 3 or a variant thereof.

[0033] (ii) the gene encoding phosphoenolpyruvate carboxylase as defined in SEQ ID NO.4

[0034] Deletion or inactivation. This can be accomplished by any means known to those skilled in the art, preferably by deleting the gene or a portion thereof, introducing at least one stop codon, or deleting or inactivating the promoter sequence. Particularly preferred is deletion of at least a portion of the protein encoding sequence of the gene (SEQ ID NO. 5).

[0035] (iii) The activity of shikimate kinase is increased. This is preferably achieved by increasing the expression of the corresponding enzyme.

[0036] In one embodiment of the present invention, the activity is increased by enhancing the expression of an endogenous shikimate kinase gene as defined by SEQ ID NO. 6 or a variant thereof. In another preferred embodiment, this is increased by expressing an exogenous shikimate kinase (preferably as defined by SEQ ID NO.

[0037] Enhanced gene expression can be achieved by any method known to those skilled in the art, in particular by introducing two or more copies of the corresponding gene into the microorganism or by using a stronger promoter for expressing endogenous enzymes. A particularly preferred promoter for expressing exogenous genes or enhancing the expression of endogenous genes is ptuf as defined in SEQ ID NO. 8.

[0038] (iv) Increased activity of 3-phosphoshikimate 1-carboxyvinyltransferase and chorismate synthase. Preferably, these enzymes have amino acid sequences as defined by SEQ ID NO. 9 or a variant thereof and SEQ ID NO. 10 or a variant thereof. This is preferably performed by introducing additional copies of the genes encoding these enzymes into the microorganism.

[0039] The ptuf promoter is preferably used to control expression.

[0040] (v) The presence of a 3-deoxyarabinoheptulosanate-7-phosphate synthase (DAHP synthase) that is "feedback-resistant," i.e., not inhibited by its product or any products formed from it. Preferably, the enzyme has the amino acid sequence defined by SEQ ID NO. 11 or a variant thereof.

[0041] In a preferred embodiment of the invention, the prokaryotic cells as defined above also exhibit increased xylose isomerase and xylulokinase activity. This increased activity is preferably achieved by enhancing the expression of both genes. This enhanced expression can be achieved by any method known to those skilled in the art, in particular by introducing two or more copies of the corresponding genes into the microorganism or by using a stronger promoter for expressing endogenous enzymes. According to the present invention, preferably the expression of an enzyme having an amino acid sequence as defined by SEQ ID NO. 12 or SEQ ID NO. 14 or a variant thereof is increased.

[0042] With respect to anthranilate phosphoribosyltransferase (SEQ ID NO. 3), shikimate kinase (SEQ ID NO. 6 or 7), 3-phosphoshikimate 1-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 present in the corresponding enzyme. The above modifications can in principle be made continuously or discontinuously at any desired site in the enzyme. However, they are preferably made only at the N-terminus and / or C-terminus of the polypeptide. Amino acid substitutions are preferably conservative substitutions, i.e. those in which the modified amino acid has a residue whose chemical properties are similar to those of the amino acid present in the unmodified enzyme. Therefore, it is more preferred to exchange amino acids with basic residues for those with basic residues, amino acids with acidic residues for those with similar 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. The enzyme specific activity of the variant is preferably at least 80% of the specific activity of the unmodified enzyme. Those skilled in the art can find enzyme tests for verifying the activity of the above enzymes in the literature.

[0043] With respect to DAHP synthase (SEQ ID NO. 11), a "variant" is understood to mean an enzyme obtained by adding, deleting, or exchanging up to 5%, preferably up to 2%, of the amino acids present in the corresponding enzyme, provided that positions 76 and 211 remain unchanged. Furthermore, positions 10, 13, 147, 148, 150, 151, 179, 209, 211, and 212 remain unchanged. In a more preferred embodiment of the invention, positions 144, 175, and 215 are also not altered in the variant. In an even more preferred embodiment of the invention, positions 92, 97, 165, 186, and 268 are also not altered in addition to the aforementioned positions. It will be understood by those skilled in the art that if amino acids are deleted or inserted, the aforementioned positions are shifted accordingly. The aforementioned modifications can, in principle, be made continuously or discontinuously at any desired site in the enzyme. However, they are preferably made only at the N-terminus and / or C-terminus of the polypeptide. Amino acid substitutions 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 unmodified enzyme. Therefore, it is more preferred to exchange amino acids with basic residues for those with basic residues, amino acids with acidic residues for those with similar 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 DAHP synthase variant also has appropriate enzymatic activity. The variant's specific enzymatic activity is more preferably at least 80% of the specific activity of the unmodified DAHP synthase according to SEQ ID NO. 11.

[0044] metabolic activity

[0045] 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 prokaryotic cells in which they take up fermentable substrates and perform biochemical conversions thereof to maintain their vital functions.

[0046] In a preferred embodiment of the present invention, metabolic activity is defined as the increase of dry biomass in the increase of cell number or aqueous composition by growth.In the case of the constant volume of aqueous composition, this means the increase of cell density / dry biomass per unit volume.In the case of volume increase (particularly by supplementing fresh substrate solution volume increase), or in the case of partially replacing aqueous composition, these changes must be considered when calculating cell number / dry biomass.Cell number / dry biomass preferably changes by the absorbance of culture medium at 600nm wavelength or measures by measuring backscattering.Measure backscattering and preferably carry out under 620nm wavelength.Herein, the conversion factor between dry biomass / cell density and absorption can be measured by method well known to those skilled in the art.In a preferred embodiment, metabolic activity is defined by the consumption of at least one fermentable substrate in aqueous composition.

[0047] The studies that form the basis of this application have shown that in the presence of a defined concentration of glycated AB, the growth rate of prokaryotic cells is increased by at least 30% compared to the presence of the same concentration of the free sodium salt of AB. Regarding the effectiveness of the aqueous compositions of the present invention, it should be noted that the AB present therein is generally not fully glycated, which means that the effectiveness may also be reduced in actual use of the compositions of the present invention.

[0048] In another preferred embodiment of the present invention, metabolic activity is defined as the production of aminobenzoic acid.

[0049] Toxicity of AB

[0050] The term "AB toxicity" refers to the adverse effects of aminobenzoic acid on prokaryotic cell metabolic activity above a specific concentration. When a specific threshold (which depends on the microorganism and culture medium used) is reached and exceeded (see WO 2019 / 073035 and WO 2019 / 073033), prokaryotic cell activity is increasingly inhibited as the AB concentration increases. In extreme cases, this can cause cell death.

[0051] In the research that forms the basis of this patent application, it has been discovered that, surprisingly, the specific toxicity of glycated AB is lower than that of free AB, i.e., a given molar concentration of free AB in the growth medium has a stronger inhibitory effect on prokaryotic cell metabolic activity than the same molar concentration of glycated AB. Therefore, it has been found that it is advantageous to provide conditions in the growth medium that promote the glycation of free AB. Because other medium components can affect AB toxicity, this comparison is preferably performed using culture media that differ only in the concentrations of free and glycated AB, and in particular, have otherwise identical sodium and ammonium ion concentrations.

[0052] Uses of aqueous compositions

[0053] In another embodiment, the present invention relates to the use of an aqueous composition as defined above for culturing prokaryotic cells.

[0054] Unless expressly stated otherwise, all definitions given above with respect to aqueous compositions also apply to this embodiment.

[0055] The term "culturing" when based on prokaryotic cells means incubating in an aqueous composition suitable for the cells, particularly a growth medium, under conditions that allow the cells to be metabolically active. This requires, in particular, that the aqueous composition contains all the components required by the cells, that the pH is maintained within the acceptable range for the specific cells, and that the temperature is maintained within an acceptable range (preferably 20°C to 40°C).

[0056] Uses of reducing sugars

[0057] In yet another embodiment, the present invention relates to the use of reducing sugars having a total sugar concentration of at least 10 g / l, preferably at least 20 g / l and more preferably at least 30 g / l for reducing the toxicity of AB.

[0058] Methods to reduce the toxicity of AB

[0059] In yet another embodiment, the present invention relates to a method for reducing the toxicity of AB, comprising the step of contacting free AB with a reducing sugar in an aqueous solution. The molar ratio of the total amount of reducing sugar aldehyde groups to the total amount of AB is at least 0.3:1.0, preferably at least 0.4:1, and even more preferably at least 0.5:1.

[0060] All definitions given above also apply to this embodiment. The definitions regarding aqueous compositions also apply to the aqueous solutions mentioned here.

[0061] Contacting can be performed by any method known to those skilled in the art. In a preferred embodiment of the present invention, the sugar is dissolved in an aqueous solution, particularly preferably in a growth medium. The growth medium preferably comprises prokaryotic cells that convert the fermentable substrate present in the growth medium to form AB. The AB formed by the prokaryotic cells is secreted by the cells and then immediately comes into contact with the reducing sugars already present in the solution.

[0062] Methods for culturing prokaryotic cells

[0063] In another preferred embodiment, the present invention relates to a method for culturing prokaryotic cells in the presence of AB and at least one reducing sugar, wherein

[0064] a) the cells are cells of the genus Corynebacterium and the total amount of aminobenzoic acid (AB) is at least 40 g / l; or

[0065] b) the cells are cells of the genus Escherichia or Pseudomonas, and the total amount of aminobenzoic acid (AB) is at least 5 g / l;

[0066] And the molar ratio of the total amount of the reducing sugar aldehyde groups to the total amount of AB is at least 0.3:1.0.

[0067] All definitions given above apply equally to this embodiment.

[0068] The prokaryotic cells are preferably cells capable of converting a fermentable substrate to form AB. Particularly preferably, they are capable of utilizing reducing sugars as fermentable substrates to achieve this purpose. Therefore, the culture method is preferably a method for forming AB with prokaryotic cells, i.e., a method for producing AB by microbial fermentation.

[0069] Incubating microbial cells in a medium in which a portion of AB is present in a saccharified form can reduce the toxicity of AB. This applies to all microbial processes in which AB is present in the medium. This primarily relates to processes for producing oAB or pAB by microbial fermentation. In such processes, an expected accumulation of AB occurs, which is beneficial to the efficiency of the process if the fermentation can be carried out for as long as possible, i.e., until the highest possible concentration of AB is reached without compromising the activity of the cells. However, reducing the toxicity of AB by saccharification is also effective in processes in which AB is not produced but the microorganism is still exposed to AB. These processes particularly refer to processes in which AB is not the intended product but rather the starting material to be converted to the target product by microbial fermentation. In general, the present invention is always advantageous when microbial cells are exposed to AB and a reduction in their metabolic activity or vitality is undesirable.

[0070] The following working examples are intended only to illustrate the present invention and are not intended to limit the scope of the claims in any way.

[0071] Working Examples

[0072] Example 1: Reaction of glucose with oAB to form gly-oAB

[0073] 150 g / L oAB was dissolved in water and adjusted to pH 7.0 by neutralization with 5 M NaOH. 120 g / L glucose was separately dissolved in water and also adjusted to pH 7.0. Nine different mixtures were prepared using varying ratios of these two solutions and water, with glucose concentrations of 10, 30, or 60 g / L and oAB concentrations of 5, 40, or 75 g / L. These mixtures were prepared in duplicate in 2 mL reaction tubes and The cells were incubated at 1000 rpm and 33°C in a Eppendorf tube (Roche). The remaining free glucose was measured using a Cedex Bioanalyzer (Roche) to track the progress of the reaction between glucose and oAB. A decrease in the measured glucose concentration (in mol / L) corresponded to an increase in the gly-oAB concentration (in mol / L). After approximately 75 hours, no further changes in glucose concentration were detected.

[0074] Table 1 shows the proportion of gly-oAB in the total amount of oAB used after 75 hours for different mixtures. Depending on the initial concentrations of glucose and oAB, approximately 8.8% to 87.3% of the total amount of oAB is present as glycated molecules at equilibrium.

[0075] Table 1: The ratio of gly-oAB to the total amount of oAB used after 75 h at different starting concentrations of glucose and oAB.

[0076]

[0077]

[0078] Example 2: Reaction of glucose with pAB to form gly-pAB

[0079] 150 g / L pAB was dissolved in water and adjusted to pH 7.0 by neutralization with 5 M NaOH. 120 g / L glucose was dissolved separately in water and also adjusted to pH 7.0. Nine different mixtures were prepared using varying ratios of these two solutions and water, with glucose concentrations of 10, 30, or 60 g / L and pAB concentrations of 5, 40, or 75 g / L. These mixtures were prepared in duplicate in 2 mL reaction tubes and The cells were incubated at 1000 rpm and 33°C in a Eppendorf tube (Roche). The remaining free glucose was measured using a Cedex Bioanalyzer (Roche) to track the progress of the reaction between glucose and pAB. A decrease in the measured glucose concentration (in mol / L) corresponded to an increase in the gly-pAB concentration (in mol / L). After approximately 75 hours, no further changes in glucose concentration were detected.

[0080] The proportion of gly-pAB in the total amount of pAB used after 75 hours for the different mixtures is shown in Table 2. Depending on the initial concentrations of glucose and pAB, approximately 7.8% to 83.3% of the total amount of pAB is present as glycated molecules at equilibrium.

[0081] Table 2: The ratio of gly-pAB to the total amount of pAB used after 75 h at different starting concentrations of glucose and pAB.

[0082]

[0083] Example 3: Determination of gly-oAB and gly-pAB by acidic reverse reaction

[0084] Due to glucose metabolism during fermentation, glycated aminobenzoic acid cannot be quantified by a decrease in glucose concentration, as this decrease could also be due to glucose consumption. However, under acidic conditions, a reverse reaction may occur from gly-oAB or gly-pAB to free glucose and aminobenzoic acid. If this reverse reaction proceeds to near completion, gly-oAB or gly-pAB can be quantified by the free glucose concentrations before and after the reverse reaction.

[0085] To verify this method, different mixtures of glucose and aminobenzoic acid described in Examples 1 and 2 were incubated for approximately 75 hours to form saccharified aminobenzoic acid. Then, 200 μL of the corresponding mixture was mixed with 800 μL of 1M hydrochloric acid and allowed to stand at room temperature. Free glucose was regularly measured using a Cedex bioanalyzer (Roche). After approximately 2 hours, no further changes in glucose concentration were detected.

[0086] The percentage of free glucose determined after the acidic reverse reaction relative to the initial glucose concentration used for the gly-pAB reverse reaction is shown in Table 3. Regardless of the concentrations of glucose and pAB used, more than 95.4% of the glucose could be recovered after the acid treatment.

[0087] Table 3: gly-pAB reverse reaction under acidic conditions

[0088]

[0089] Table 4 shows the percentage of free glucose measured after the acidic reverse reaction relative to the initial glucose concentration used for the gly-oAB reverse reaction. As the oAB concentration increases and the glucose concentration decreases, the proportion of glucose present that can be recovered after acid treatment decreases. However, when the glucose concentration is 30 g / L or higher and the oAB concentration is up to 75 g / L, greater than 88.2% of the glucose present can be recovered. Therefore, the acidic reverse reaction can be used to quantify gly-pAB and, under defined conditions, gly-oAB.

[0090] Table 4: gly-oAB reverse reaction under acidic conditions

[0091]

[0092] Example 4: Demonstration of reduced toxicity of saccharified oAB or pAB to microorganisms

[0093] General Notes on Experimental Design

[0094] Following embodiment uses the BioLector Pro micro-culture system from m2p laboratory (Aachen, Germany), and this system can carry out 48 parallel cultures in a microtiter plate.Bacterial strain is cultivated under 1000rpm (3mm amplitude) in initial 800 μ L and final 1500 μ L culture medium in BOH2 48-hole flower plate (Flowerplate) (m2p laboratory, Aachen, Germany).Used plate contains immobilized indicator material (optical electrode (optode)), can measure pH value and oxygen partial pressure (pO2) in each hole online.At 620nm place, by measuring scattered light signal (gain is set to 3) observation all cultures' growth.The parallel culture system from m2p laboratory is known and is applicable to cultivating bacterial strain.

[0095] Cultivation temperature and the substratum used depend on the bacterial strain to be tested, and together with other cultivation parameters are listed in detail in Table 5.Example Select wild-type bacterial strain Corynebacterium glutamicum ATCC 13032, intestinal bacteria (Escherichiacoli) K12 and Pseudomonas putida (Pseudomonas putida) DSM 6125.In main cultivation, Corynebacterium glutamicum uses CG XII minimal medium, intestinal bacteria and Pseudomonas putida use LB complex medium (Miller's LB broth (L3522, Sigma-Aldrich, St.Louis, USA)), these substratums are all the cultures that are generally used for these bacterial species and related genera.The precise substratum composition with adjustment is listed in detail in Table 6.

[0096] Table 5: Pre-culture, culture medium and culture parameters of the following examples

[0097]

[0098]

[0099] Table 6: Culture medium composition and stock solutions

[0100]

[0101] To determine the toxic effects of free oAB or free pAB compared to the glycosylated forms gly-oAB or gly-pAB, the main culture medium containing various concentrations of oAB and gly-oAB or pAB and gly-pAB was further added after the initial early growth phase (hereinafter referred to as the spike group). The main culture medium containing the gly-oAB or gly-pAB forms was prepared according to the kinetics of Examples 1 and 2 and stirred for 70 hours. As a reference, a medium without AB was added (hereinafter referred to as the reference spike group).

[0102] Because the addition of gly-oAB or gly-pAB to the culture medium results in an increase in the amount of carbon supplied in the form of complex-bound glucose and free glucose remaining in the equilibrium reaction, an equal amount of glucose was also supplied in the reference spike group and in the oAB and pAB spike groups, but without prior incubation. This means that the oAB or pAB complex with glucose had not yet formed at the time of addition. This was experimentally verified using the method described in Example 3.

[0103] Table 7 (oAB) and Table 8 (pAB) provide an overview of the experimental setup. For each setup, the types and amounts of the various solutions added after the early growth phase are listed. The amounts are in each case based on a single culture well in a microtiter plate. Although 800 μL of inoculum medium was used at the start of the culture, a total of 700 μL of solution was added to each well after the early growth phase. Furthermore, the corresponding AB concentrations obtained after spike addition and the increase in glucose concentration in the wells due to spike addition are clearly stated. Table 6 also describes the composition and preparation of the solutions used.

[0104]

[0105]

[0106]

[0107]

[0108] Example 4.1: Toxicity of gly-oAB and free oAB to Corynebacterium glutamicum ATCC 13032

[0109] For the following examples, the strain Corynebacterium glutamicum ATCC 13032 was cultured in 800 μL of CG XII minimal medium in a BioLector Pro microculture system from m2p Laboratories as described above. After 7.2 hours, oAB and gly-oAB at different concentrations were added. All settings and references were performed in triplicate (Table 7: Example 4.1).

[0110] The toxicity of oAB and gly-oAB was investigated based on growth in comparison to two unspiked controls (Reference 1 and Reference 2). To this end, the slope of the scattered light signal was analyzed within the first 10 hours after spiking (Table 9). Both oAB and gly-oAB caused growth retardation. However, gly-oAB was found to be less toxic than free oAB at all concentrations, based on the larger slope of the scattered light signal, at the same oAB concentration in the oAB or gly-oAB spiked groups.

[0111] Table 9: Slope of the scattered light signal of a culture of the strain Corynebacterium glutamicum ATCC 13032 during the first 10 hours after fortification with oAB, gly-oAB or a mixture of culture medium and glucose solution (reference).

[0112]

[0113] Example 4.2: Toxicity of gly-oAB and free oAB to Escherichia coli K12 and Pseudomonas putida DSM 6125

[0114] For the following examples, the strains E. coli K12 and P. putida DSM 6125 were cultured as described above in a BioLector Pro microculture system from m2p Laboratories in 800 μL of LB medium. After 4 hours (E. coli) and 7 hours (P. putida), different concentrations of oAB and gly-oAB were added. All setups and references were performed in triplicate (Table 7: Example 4.2).

[0115] Tables 10 and 11 show the slope of the scattered light signal for E. coli within the first 2 hours after spiking and the slope of the scattered light signal for Pseudomonas putida within the first 10 hours after spiking, respectively. In both microorganisms, oAB and gly-oAB had growth inhibitory / toxic effects compared to the reference. In the case of E. coli, at the lowest oAB concentration (5 g / L), the growth impairment caused by oAB and gly-oAB was similar. However, at 10 g / L and 15 g / L oAB, gly-oAB was found to have lower toxicity than free oAB based on the greater slope of the scattered light signal. In the case of Pseudomonas putida, gly-oAB was found to have lower toxicity than free oAB based on the slope of the scattered light signal even at the lowest concentration (5 g / L). At 10 g / L free oAB, growth did not occur after spiking, while at the same concentration of gly-oAB, cells continued to grow. At the highest concentration (15 g / L), no growth occurred after spiking, neither in the case of free oAB nor in the case of gly-oAB.

[0116] Therefore, it could be demonstrated that the saccharification of oAB reduced the toxic effects not only on Corynebacterium glutamicum ATCC 13032, but also on Escherichia coli K12 and Pseudomonas putida DSM 6125.

[0117] Table 10: Slope of the scattered light signal of cultures of the strain E. coli K12 during the first 2 hours after fortification with oAB, gly-oAB or a mixture of culture medium and glucose solution (reference).

[0118]

[0119] Table 11: Slope of the scattered light signal of a culture of the strain Pseudomonas putida DSM 6125 during the first 10 hours after fortification with oAB, gly-oAB or a mixture of culture medium and glucose solution (reference).

[0120]

[0121] Example 4.3: Toxicity of gly-pAB and free pAB to Corynebacterium glutamicum ATCC 13032, Escherichia coli K12, and Pseudomonas putida DSM 6125

[0122] The toxicity of pAB and gly-pAB was investigated as described above for oAB and gly-oAB in Examples 4.1 and 4.2 and shown in Table 8. The results are summarized in Tables 12, 13 and 14. Here, the slope of the scattered light signal after spiking is shown for all three strains.

[0123] In all cases, it was apparent that the addition of free pAB or gly-pAB caused a growth impairment compared to the reference culture without pAB addition. For C. glutamicum ATCC 13032, gly-pAB was found to have reduced toxicity compared to the free form of pAB in all cases, based on the slope of the scattered light signal (Table 12), similar to the data previously described for gly-oAB and oAB (Table 9).

[0124] Table 12: Slope of the scattered light signal of a culture of the strain Corynebacterium glutamicum ATCC 13032 during the first 4.7 hours after fortification with pAB, gly-pAB or a mixture of culture medium and glucose solution (reference).

[0125]

[0126]

[0127] For E. coli K12, no advantage was observed for gly-pAB compared to the free form of pAB after 4 hours of addition of 5 g / L pAB. Because glucose in the pAB-spiked group is unbound and thus accessible to the microorganisms for use as a carbon source, the pAB-spiked group exhibited a larger light scattering signal slope compared to the gly-pAB-spiked group. The larger light scattering signal slopes at 10 g / L and 15 g / L indicate that the glycated form exhibited a lower toxic effect compared to the pAB-spiked group.

[0128] Table 13: Slope of the scattered light signal of cultures of the strain E. coli K12 during the first 4 hours after fortification with pAB, gly-pAB or a mixture of culture medium and glucose solution (reference).

[0129]

[0130] For Pseudomonas putida DSM 6125, no difference was observed between pAB and gly-pAB 5 hours after addition of 5 and 10 g / L pAB. However, at 15 g / L, the glycated form exhibited a lower toxic effect compared to the pAB spiked group, as evident by the larger slope of the scattered light signal.

[0131] Table 14: Slope of the scattered light signal of a culture of the strain Pseudomonas putida DSM 6125 during the first 8 hours after fortification with pAB, gly-pAB or a mixture of culture medium and glucose solution (reference).

[0132]

Claims

1. An aqueous composition comprising a) anthranilic acid (oAB) in a total amount of at least 40 g / l and a plurality of cells of the genus Corynebacterium; or b) anthranilic acid (oAB) in a total amount of at least 5 g / l and a plurality of cells of the genus Escherichia or Pseudomonas; wherein at least 2 g / l of the total amount is in the form of glycated AB (gly-AB).

2. The aqueous composition of claim 1, wherein at least 15% of the total amount of oAB is in the form of saccharified oAB.

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

0.

4. The aqueous composition according to claim 3, comprising at least one reducing sugar which cannot be used by cells present 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, further comprising a nitrogen source available to the cells, a sulfur source available to the cells present, and a phosphorus source available to the cells present.

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

7. The aqueous composition of 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 culturing cells present therein.

9. A method for reducing the toxicity of aminobenzoic acid (AB), the method comprising the step of contacting free AB with a reducing sugar in an aqueous solution, wherein the molar ratio of the total amount of reducing sugar aldehyde groups to the total amount of 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 cells present in the aqueous composition.

11. Use of reducing sugars having a total sugar concentration of at least 10 g / l for reducing the toxicity of AB.

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

0.

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

0.

14. The method of claim 13, wherein the cell is capable of converting a fermentable substrate to form oAB.

15. The method of claim 14, wherein the cells are capable of utilizing reducing sugars as fermentable substrates.

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

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