Tolerance of microbial cells to anthranilic acid in the presence of alkali metal ions at neutral pH

Abstract

The present invention relates to the production of anthranilic acid from a fermentable substrate using microbial cells and an alkali metal-containing base during fermentation.

Description

[0001] The present invention relates to the production of anthranilic acid from a fermentable substrate using microbial cells and an alkali metal-containing base during fermentation.

[0002] At present, there is no renewable or biologically derived source of commercially available anthranilate or corresponding acid. Current aniline production methods rely on the chemical synthesis of petroleum-derived raw materials. Contrary to renewable raw materials such as renewable resources "biomass", this petroleum-derived raw material is non-renewable. The chemical synthesis of aniline is a multi-step process. Several reaction steps involved in the production of aniline lead to high production costs. In addition, conventional (i.e. chemical) aniline synthesis is related to dangerous intermediates, solvents and waste products, which can have a significant impact on the environment. Non-specific side reactions on the aromatic ring lead to a reduction in product yield, thus further increasing production costs. Petroleum-derived raw materials are subject to the impact of cost fluctuations caused by global oil prices.

[0003] Anthranilic acid and / or salts (o-aminobenzoate) are natural intermediates in the shikimate acid pathway and are precursors for the biosynthesis of the aromatic amino acid L-tryptophan. WO2015 / 124687 discloses a concept for producing biologically derived aniline in two process steps: (1) fermentative production of anthranilic acid and / or salts (o-aminobenzoate) using recombinant bacteria and (2) subsequent catalytic conversion of anthranilic acid into aniline. The recombinant bacteria used in the process belong to Corynebacterium ( Corynebacterium ) or Pseudomonas ( Pseudomonas ) family. Both bacteria produce anthranilic acid and / or salts at pH values ​​between 7 and 8.

[0004] When producing o-aminobenzoic acid and / or salts at pH 7 to pH 8, there are the following problems: Since fermentation produces o-aminobenzoate, which is an acid, a base such as NH4OH needs to be added to ensure a stable neutral pH. + / Anthranilate - However, such anthranilates are toxic to microbial cells. Figure 3 When the NH4 content reaches greater than 25 g / L o-aminobenzoate + At concentrations of 50 g / L / o-aminobenzoate, the metabolic activity of the bacterial cells (see OTR) is limited, and cell growth (see dry weight) is arrested at higher concentrations (>50 g / L). This toxicity is unknown for other products such as glutamate or lysine.

[0005] This type of toxicity problem is usually solved by direct evolution of the applied microbial cells. First, in repeated batch experiments or continuous fermentation experiments, the microbial cells are exposed to increasing concentrations of toxic components (e.g., anthranilic acid and / or salts). Thus, the microbial cells evolve by random mutagenesis (which can be accelerated by adding mutagens), and more tolerant microbial cells survive. Secondly, the most tolerant cells are isolated / selected and can be used for production.

[0006] However, many of the mechanisms that underlie tolerance in these microbial cells consume energy (Aindrila Mukhopadhyay, Trends in Microbiology , Vol. 23, No. 8, August 2015; Rau et al., Microb Cell Fact (2016) 15: 176; Warnecke T, Gill RT. Microbial Cell Factories .2005; 4: 25). Therefore, a certain proportion of fermentable substrates is consumed to maintain metabolism, resulting in a reduction in the yield of anthranilic acid and / or salt. For this reason, the high titer of anthranilic acid and / or salt required for the reasonable space-time yield of the process concomitantly reduces the product yield.

[0007] Although biotechnological production of anthranilic acid and / or salts from renewable sources as precursors for aniline production offers potential benefits, the above-mentioned toxicity factors reduce the potential benefits of this process. Therefore, alternative methods for increasing the tolerance of microbial cells to anthranilic acid are needed.

[0008] The problem is solved by the embodiments defined in the claims and in the following description.

[0009] In a first embodiment, the present invention relates to a method for culturing microbial cells in the presence of at least 30 g / l of anthranilic acid and / or salts (oAB), the method comprising the steps of adding alkali metal ions to the culture medium such that the molar ratio of alkali metal ions to oAB is in the range of 0.75 to 1.25.

[0010] The microbial cells are, preferably, cells capable of bioconverting a fermentable substrate into oAB. The term "bioconversion" refers to a biochemical process that converts one or more molecules of a fermentable substrate into one or more molecules of oAB. These processes are primarily mediated by enzymes expressed by bacterial cells.

[0011] The term "o-aminobenzoic acid" (or oAB) as referred to in this application relates to 2-aminobenzoic acid. This compound is also known as anthranilic acid. The skilled person knows that the acid can exist in its protonated form as a neutral substance or deprotonated as an anion. In aqueous solution, part of the acid is protonated and part is present as an anion. The ratio between the protonated acid and the anion depends on the pH of the solution and the dissociation constant K of the acid in question. a Unless otherwise stated, the term "anthranilic acid" as used in this application always refers to both the protonated acid and the corresponding anion.

[0012] The term "medium" is generally understood in the art. It refers to an aqueous solution that provides conditions that allow the metabolic activity of microbial cells. The conditions are physicochemical, such as temperature, dissolved oxygen concentration, ionic strength and pH. They are also chemical and include the concentrations of different nutrients that the microbial cells need for their activity. Those skilled in the art can adapt these conditions to the needs of specific microbial cells based on the common knowledge available for the specific microbial cells.

[0013] The microbial cells used in the present invention may be naturally occurring strains, i.e. microbial strains that are capable of converting fermentable substrates into oAB without any further human interaction (especially without genetic manipulation). However, in a preferred embodiment of the present invention, they are microbial cells that have acquired the above-mentioned capabilities during genetic manipulation or microbial cells that have used these methods to improve pre-existing capabilities.

[0014] The term "genetic modification" within the meaning of the present invention refers to the change in the nucleotide sequence of a given gene of a microbial host compared to the wild-type sequence. This genetic modification can include the disappearance and insertion of one or more deoxyribonucleic acids. This genetic modification can include partial or complete disappearance and the insertion introduced into the genome of the microbial host by transformation. This genetic modification can produce a recombinant microbial host, wherein the genetic modification can include the change of at least one, two, three, four or more mononucleotides compared to the wild-type sequence of the microbial host respectively. For example, the genetic modification can be to disappear or insert at least one, two, three, four or more mononucleotides, or to convert at least one, two, three, four or more mononucleotides. Genetic modification according to the present invention can have the effect of for example reducing the expression of a gene respectively or for example enhancing the expression of a gene respectively.

[0015] The microbial cell is a prokaryotic cell or a eukaryotic cell. Preferably, the prokaryotic cell is a bacterial cell. Preferably, the bacterial cell belongs to the genus Corynebacterium, Mycobacterium ( Mycobacterium ) genus, Bacillus genus ( Bacillus), Pseudomonas, Escherichia ( Escherichia ) and Vibrio ( Vibrio ) genus. More preferably, Corynebacterium glutamicum ( Corynebacterium glutamicum ) and Pseudomonas putida ( Pseudomonas putida ). Most preferably, Corynebacterium glutamicum ATCC 13032. Preferred eukaryotic cells belong to yeast ( Saccharomycetales ) or Aspergillus ( Aspergillus More preferably, they belong to the genus Ashbya gossypii ( Ashbya gossypii ) species, Pichia pastoris ( Pichia pastoris ) species, Hansenula polymorpha ( Hansenula polymorpha ) species, Yarrowia lipolytica ( Yarrowia lipolytica ) species, Zygosaccharomyces bailii ( Zygosaccharomyces bailii ) species, Kluyveromyces marxianus ( Kluyveromyces marxianus ) species and Saccharomyces cerevisiae ( Saccharomyces cerevisiae Most preferably, the yeast is Saccharomyces cerevisiae.

[0016] Particularly preferably, the microbial cell is characterized by dP Genetic modification of a gene that prevents or reduces the expression of the gene and / or that results in a gene product having reduced or no enzymatic activity. Such microbial cells can be readily produced by one of ordinary skill in the art using conventional genetic methods.

[0017] Recombinant bacterial cells particularly suitable for use in the methods of the invention are disclosed in WO 2015 / 124687.

[0018] The term "culture" refers to the cultivation of microbial cells under conditions that promote metabolic activity. These conditions are known to those skilled in the art. The conditions include at least the presence of microbial cells in a medium suitable for cell growth at a temperature that allows cell proliferation, the presence of a fermentable substrate, and the presence of oxygen. Preferably, the metabolic activity is oxygen consumption. More preferably, the metabolic activity is cell proliferation measured by an increase in dry weight. Most preferably, the metabolic activity is the bioconversion of a fermentable substrate to produce oAB.

[0019] Preferably, the culturing is carried out in a medium having a pH of 6.0-8.0. Preferably, the pH is maintained within this range, as described below in this application.

[0020] Fermentable substrate as understood by the application is any organic compound or mixture of organic compounds, which can be utilized by microbial cells to produce anthranilic acid in the presence or absence of oxygen.Preferred fermentable substrates also serve as energy sources and carbon sources for microbial cell growth.Preferred fermentable substrates are processed beets, sugar cane, starchy plants and lignocellulose.Also preferred as fermentable substrates are glycerol and C1 compounds, preferably CO, and fermentable sugars.Preferred fermentable sugars are glucose.

[0021] Suitable alkali metal ions for the process according to the invention are all ions of alkali metals. Preferred alkali metals are sodium, potassium and rubidium. Particularly preferred alkali metals are sodium. According to the invention, mixtures of at least two different alkali metal ions can also be used.

[0022] Preferably, alkali metal ions are added to the culture medium as a component of the base comprising said ions. In the present invention, such a base is referred to as an "alkali base". More preferably, the alkali base is a hydroxide. Alternative alkali-containing bases include carbonates or phosphates such as disodium phosphate. It should be understood that the term "alkali base" also refers to a mixture of at least two different alkali bases.

[0023] In another embodiment, the present invention relates to a method for producing oAB comprising the steps of:

[0024] a) cultivating microbial cells in the presence of a fermentable substrate and under conditions suitable for bioconversion of the fermentable substrate to oAB; and

[0025] b) Adding an alkali metal base to the fermentation broth as a buffer substance.

[0026] All definitions given above also apply to this embodiment.

[0027] Those skilled in the art can select cultivation conditions suitable for bioconversion of fermentable substrates to oAB based on the known properties of the microbial cells and the cultivation requirements. Whether the cultivation conditions are suitable for the production of oAB can be easily determined by measuring the concentration of oAB in the culture medium. An increase in the concentration of oAB over time indicates that the cultivation conditions meet the requirements.

[0028] "Fermentable substrate" is any carbon compound that can be converted into oAB by microbial cells. Typically, the fermentable substrate will also provide nutrients (feed) for the maintenance and / or growth metabolism of the microbial cells. However, in special cases, it may be necessary to supplement the fermentable substrate by providing additional substrates that provide nutrients for the maintenance and / or growth metabolism of the microbial cells. Preferred fermentable substrates are selected from C-5 monosaccharides, C-6 monosaccharides, disaccharides and trisaccharides. The C-5 monosaccharides are, preferably, xylose and arabinose. The C-6 monosaccharides are, preferably, glucose, fructose or mannose. The disaccharides are, preferably, sucrose. The trisaccharides are, preferably, sucrose trisaccharides.

[0029] Preferably, the dry weight of the microorganisms at least doubles during the cultivation process. More preferably, a dry weight of at least 6 g / l is reached at the end of the cultivation.

[0030] Since oAB is an acid, base needs to be added to keep the pH of the culture medium stable. However, apart from its effect on pH (which can be mitigated by adding base), above a certain concentration, oAB itself is toxic to microorganisms. In the studies underlying the present invention, it has surprisingly been found that the same bacterial strain tolerates higher oAB concentrations if the base used to maintain a stable pH contains an alkali metal ion as a cation.

[0031] The total amount of all base added in process step b) depends on the amount of oAB produced during the time of base addition. It must be sufficient to prevent the pH from falling too much without causing the pH to rise beyond the range tolerated by the microorganism in question. Preferably, the pH is maintained by adding base in a range in which the metabolic activity of the microorganism in question is at least 60%, more preferably at least 80%, of the activity at the optimal pH. All definitions of "metabolic activity" given above also apply here. Therefore, the time and amount of base addition are preferably based on the measurement of the pH in the culture medium, as is commonly practiced in industrial biotechnology. The addition can be carried out in a continuous manner as a steady stream. It can also be carried out by adding discrete doses of base at different time points.

[0032] From the above discussion, therefore, method step b) is carried out simultaneously with method step a), that is, the sodium-containing base is added while the microorganism cultivation is being carried out.

[0033] In the presence of an alkali metal base, in particular a sodium-containing base, the microbial cell as defined above tolerates at least 200%, more preferably at least 170%, even more preferably at least 150% and most preferably at least 130% of the oAB concentration tolerated by the same microbial cell in the absence of an alkali metal base and under otherwise identical culture conditions.

[0034] Preferably, the minimum concentration of oAB tolerated is 30 g / l, more preferably 40 g / l, and most preferably 50 g / l.

[0035] According to the invention, the base added in process step b) need not consist exclusively of alkali metal bases. The invention also envisages the use of a mixture of alkali metal bases and other bases in process step b). "Other bases" are, for example, gaseous ammonium, ammonium hydroxide, calcium hydroxide or calcium carbonate. However, it is preferred that the majority of the base added in process step b) is a sodium-containing base. In this case, it is important to note that other bases, in particular those containing nitrogen, may be consumed by the microbial cells. Therefore, preferably, the ratio of alkali metal base and "other base" is defined not by the amount added but by the molar ratio of the alkali metal and other bases actually present in the culture medium at a given point in time. Preferably, the addition of alkali metal base and other bases in process step b) is carried out in such a way that the molar amount of alkali metal base accounts for at least 30 mol-%, more preferably at least 50 mol-%, even more preferably at least 75 mol%, and most preferably at least 90 mol%. It is preferred to maintain these limits throughout the incubation time. However, for practical reasons, it is acceptable that the amount of alkali metal base falls below the above-defined value as long as the above-defined molar ratio is maintained during at least 90% of the duration of the total incubation time.

[0036] The aforementioned percentages refer to the amount of cations. In the most preferred embodiment, the base added in process step b) contains no cations other than sodium within the purity range of reagents commonly used for large-scale fermentations. A typical sodium-containing base is NaOH, which is a by-product of the chlor-alkali process. The concentration of the base is about 50% by weight NaOH.

[0037] In a particularly preferred embodiment of the present invention, it is defined as NH3 and NH4 in the culture medium. + The amount of ammonium in the sum of the concentrations of does not exceed 300 mM, preferably 200 mM, more preferably 100 mM, even more preferably 50 mM.

[0038] A microbial cell is "tolerant" to a given amount of oAB if its metabolic activity as defined above in the present application is not reduced by more than 50%, more preferably by more than 25%, compared to the activity exhibited in the absence of oAB. It will be appreciated that any sudden increase in the concentration of oAB causes a transient decrease in the metabolic activity of the microbial cell (see the Examples). During the oAB production process, this effect is unlikely to be encountered because the oAB concentration is gradually increased, allowing the microbial cells time to adapt. However, the addition of oAB for testing purposes may have this effect. Therefore, it is preferred to measure the metabolic activity of the microbial cell at least two hours after any sharp increase in the oAB concentration (e.g. caused by the addition of oAB to the culture medium). Otherwise, for given conditions, the true lasting effect of oAB on activity may be overestimated.

[0039] If oAB is produced by microbial cells under the conditions in question, it accumulates in the microbial cells and / or in the culture medium. A person skilled in the art is fully aware of a variety of methods suitable for recovering the desired product from the cells or culture medium. Preferred methods are disclosed in WO 2015 / 124687.

[0040] Advantageously, the use of an alkali metal base allows a given microbial strain to be grown in the presence of higher concentrations of oAB than with other bases. Thus, an increase in oAB tolerance can be achieved without the time-consuming process of new strain development. Furthermore, many of the mechanisms underlying oAB tolerance of a particular engineered strain consume energy, so less fermentable substrate is available for actual conversion to oAB, resulting in reduced substrate yield.

[0041] In another embodiment, the present invention relates to the use of an alkali metal base as a buffer substance during the bioconversion of a fermentable substrate to oAB by microbial cells.

[0042] In yet another embodiment, the present invention relates to the use of alkali metal ions to increase the tolerance of microbial cells to oAB. The alkali metal ions are preferably sodium or potassium ions.

[0043] In yet another embodiment, the present invention relates to the use of an alkali metal base to increase the tolerance of microbial cells to oAB.

[0044] The following examples are intended only to illustrate the present invention. They should not limit the scope of the claims in any way.

[0045] Example

[0046] The accompanying drawings show:

[0047] Figure 1: Evaluation of the effects of modified Corynebacterium glutamicum ( C. glutamicum) Maximum tolerable oAB concentration of the production strain during batch cultivation in a 1 L bench-scale bioreactor. 20 mL of 500 g / L oAB stock solution (oAB dissolved in NaOH at pH 7) was added after 23 and 39 h, 48 h (twice), 64 h (twice) and 71 h (twice), and glucose solution was added after 40 h, 48 h, 63 h, 65 and 71.5 h to prevent glucose limitation. Initial culture volume VL = 1 L, temperature 30 °C, pH = 7 controlled by adding NH4OH (10 w% NH3) during cultivation, air aeration rate = 0.2 L / min, pO2 was controlled at 30% air saturation by adjusting the stirrer speed between 200 and 1200 rpm.

[0048] Figure 2: Effect of the addition of Na-oAB and NH4-oAB on oAB tolerance during batch culture at 1L scale with Corynebacterium glutamicum ATCC 13032. Filled symbols: 40 mL of 500 g / L oAB stock solution (oAB dissolved with NaOH at pH 7) was added after 7 hours and 24 hours. Open symbols: 40 mL of 500 g / L oAB stock solution (oAB dissolved with NH4OH at pH 7) was added after 7 hours and 24 hours. Two bioreactors: 36 g / L glucose (added as stock solution) was added after 7.5 hours and 24.5 hours to prevent glucose limitation. Initial culture volume VL = 1L, temperature 30°C, pH = 7 controlled with NH4OH (10 w% NH3), air aeration rate = 0.2L / min, pO2 was controlled at 30% air saturation by adjusting the agitator speed at 200 to 1200 rpm.

[0049] Figure 3: Effect of the addition of Na-oAB and NH4-oAB on oAB tolerance during batch cultivation at 1 L scale with Corynebacterium glutamicum ATCC 13032. Closed symbols: 40 mL of 500 g / L oAB stock solution (oAB dissolved with NaOH at pH 7) was added after 6.4 h and 23.6 h. Open symbols: 40 mL of 500 g / L oAB stock solution (oAB dissolved with NH4OH at pH 7) was added after 6.4 h and 23.6 h. Initial culture volume VL = 1 L, temperature 30 °C, pH = 7 controlled with NH4OH (10 w% NH3), air aeration rate = 0.2 L / min, pO2 was controlled at 30% air saturation by adjusting the stirrer speed between 200 and 1200 rpm.

[0050] Example 1: Evaluation of the maximum tolerable oAB concentration of modified C. glutamicum production strains.

[0051] With modified Corynebacterium glutamicum production strain The maximum tolerable oAB concentration in the presence of sodium ions in the culture medium was tested. This strain was derived by evolutionary engineering from Corynebacterium glutamicum ATCC 13032 to increase its tolerance to oAB. The strain was then genetically modified to enable oAB production. The modifications incorporated had the following effects: reduced expression of the trpD gene encoding anthranilate phosphoribosyltransferase, knockout of the gene ppc encoding PEP carboxylase, constitutive overexpression of heterologous genes encoding feedback-tolerant DAHP synthase and anthranilate synthase, respectively, from Escherichia coli. aroG D146N and trpEG S40F Gene, constitutively overexpressing the gene aroL encoding shikimate kinase from Escherichia coli.

[0052] A bioreactor with a nominal volume of 1 L was filled with sterile culture medium consisting of an initial amount of 20 g / L glucose, 5 g / L (NH4)2SO4, 1 g / L KH2PO4, 1 g / L K2HPO4, 0.25 g / L MgSO4·7 H2O, 0.01 g / L CaCl2·2 H2O, 2 mg / L biotin (vitamin B7), 0.03 g / L protocatechuic acid (3,4-dihydroxybenzoic acid), 0.01 g / L MnSO4·H2O, 0.01 g / L FeSO4·7H2O, 1 mg / L ZnSO4·7H2O, 0.2 mg / L CuSO4·5H2O, and 0.02 mg / L NiCl2·6H2O.

[0053] The preculture medium for cultivation in shake flasks additionally contained 42 g / L MOPS buffer, 3.7 g / L brain heart infusion broth, 5 g / L urea (CH4N2O), and 20 g / L (NH4)2SO4 (instead of 5 g / L). The preculture was cultivated in 300 mL shake flasks with a liquid volume of 25 mL at a temperature of 28°C and a shaking frequency of 180 rpm until an OD of 600 > 20.

[0054] The cultivation was carried out in a laboratory-scale bioreactor with an initial culture volume of 1 L. During the fermentation, the temperature was controlled at 30 °C and the pH was kept constant at pH = 7 by adding NH4OH aqueous solution (10 w% NH3). The aeration rate was adjusted to 0.2 L / min air and the dissolved oxygen tension was controlled at 30% air saturation by controlling the agitator speed between 200 rpm and 1200 rpm. The cultivation results are shown in Figure 1 oAB concentration was increased stepwise by adding 20 mL of 500 g / L oAB stock solution (oAB dissolved in NaOH at pH 7) after 23 h, 39 h, 48 h (twice), 64 h (twice), and 71 h (twice). Glucose was added as a stock solution after 40 h, 48 h, 63 h, 65, and 71.5 h to prevent glucose limitation. Figure 1 As shown in Figure 1, an increase in biomass concentration was observed even at an oAB concentration of 80 g / L. An increase in oAB concentration from 80 g / L to 100 g / L after 71 hours resulted in a decrease in metabolic activity (indicated by a decreasing OTR signal), and no further increase in biomass was observed at this time. This experiment demonstrated that the addition of oAB as a sodium salt to the bioreactor in the presence of 80 g / L oAB could be achieved. Corynebacterium glutamicum production strain growth.

[0055] Example 2: Comparison Corynebacterium glutamicum Metabolic activity of ATCC 13032 after addition of NH4-oAB and Na-oAB during cultivation.

[0056] A Corynebacterium glutamicum strain derived from ATCC 13032 was used to compare the metabolic activity after adding NH4-oAB and Na-oAB during the culture period. This strain was derived from Corynebacterium glutamicum ATCC 13032 by evolutionary engineering to increase its tolerance to oAB. It was not further genetically modified. Therefore, it cannot produce oAB. For this purpose, two 1 L bioreactors were filled with sterile culture medium including the following initial concentrations: 20 g / L glucose, 5 g / L (NH4)2SO4, 1 g / LKH2PO4, 1 g / L K2HPO4, 0.25 g / L MgSO4·7 H2O, 0.01 g / L CaCl2·2 H2O, 2 mg / L biotin (vitamin B7), 0.03 g / L protocatechuic acid (3,4-dihydroxybenzoic acid), 0.01 g / L MnSO4·H2O, 0.01 g / L FeSO4·7H2O, 1 mg / L ZnSO4·7H2O, 0.2 mg / L CuSO4·5H2O, and 0.02 mg / L NiCl2·6H2O.

[0057] The preculture medium for cultivation in shake flasks additionally contained 42 g / L MOPS buffer, 3.7 g / L brain heart infusion broth, 5 g / L urea (CH4N2O), and 20 g / L (NH4)2SO4 (instead of 5 g / L). The preculture was cultivated in 300 mL shake flasks with a liquid volume of 25 mL at a temperature of 28°C and a shaking frequency of 180 rpm until an OD of 600 > 20.

[0058] The results of dry biomass, oAB and NH4 concentrations and oxygen transfer rate (OTR) signals are shown in Figure 2 After adding 40 mL of 500 g / L oAB stock solution (oAB dissolved with NaOH at pH 7) to Reactor 1 at 7 and 24 h of culture time, the biomass concentration and OTR signal continued to increase ( Figure 2 In contrast, the addition of NH4-oAB to Reactor 2 (added by injecting 40 mL of a 500 g / L oAB stock solution containing oAB dissolved in NH4OH at pH 7) resulted in a constant OTR signal and reduced biomass accumulation after 24 h ( Figure 2 The experiment shows that the use of Na + Replacement of NH4 + As the counter ion of oAB, it improves Corynebacterium glutamicum Tolerance of ATCC 13032 to oAB.

[0059] Example 3: Comparison Corynebacterium glutamicum Metabolic activity of ATCC 13032 after addition of NH4-oAB and Na-oAB during culture

[0060] use Corynebacterium glutamicumATCC 13032 (without further genetic modifications, not modified by evolutionary engineering) was tested for the effects of NH4 using two 1-L bioreactors filled with sterile medium containing the following initial concentrations: 20 g / L glucose, 5 g / L (NH4)2SO4, 1 g / L KH2PO4, 1 g / L K2HPO4, 0.25 g / L MgSO4·7 H2O, 0.01 g / L CaCl2·2 H2O, 2 mg / L biotin (vitamin B7), 0.03 g / L protocatechuic acid (3,4-dihydroxybenzoic acid), 0.01 g / L MnSO4·H2O, 0.01 g / L FeSO4·7H2O, 1 mg / L ZnSO4·7H2O, 0.2 mg / L CuSO4·5H2O, and 0.02 mg / L NiCl2·6H2O.

[0061] The preculture medium for cultivation in shake flasks additionally contained 42 g / L MOPS buffer, 3.7 g / L brain heart infusion broth, 5 g / L urea (CH4N2O), and 20 g / L (NH4)2SO4 (instead of 5 g / L). The preculture was cultivated in 300 mL shake flasks with a liquid volume of 25 mL at a temperature of 28°C and a shaking frequency of 180 rpm until an OD of 600 > 20.

[0062] During fermentation, the temperature was controlled at 30°C and the pH was kept constant at pH = 7 by adding NH4OH aqueous solution (10 w% NH3). The aeration rate was adjusted to 0.2 L / min air and the dissolved oxygen tension was controlled at 30% air saturation by controlling the stirrer speed between 200 rpm and 1200 rpm.

[0063] The results of dry biomass and oAB concentration and the related signal of oxygen transfer rate (OTR) are shown in Figure 3 After 6.4 hours and 23.6 hours, 40 mL of 500 g / L oAB stock solution (oAB dissolved in NaOH at pH 7) was added to Reactor 1 (closed symbols), and after 6.4 hours and 23.6 hours, 40 mL of 500 g / L oAB stock solution (oAB dissolved in NH4OH at pH 7) was added to Reactor 2 (open symbols)

[0064] like Figure 3 As shown in Figure 1, after adding Na-oAB to Reactor 1, biomass continued to accumulate ( Figure 3 In contrast, the addition of NH4-oAB to Reactor 2 caused growth inhibition after 24 hours ( Figure 3 The hollow symbols in the figure show that, compared with the case of high NH4 + Compared with the toxicity in the presence of high Na + oAB in the presence of concentration Corynebacterium glutamicum The toxicity of ATCC13032 was reduced.

[0065] The above effect can be extended at least to the genus Corynebacterium. Strains subjected to evolutionary engineering (a method of changing the genetic material of an organism in a random manner) showed the same behavior as the strain used in Example 3. Therefore, the effect seems to be based on some rather basic functions of the cell that are not easily changed.

Claims

1. A method for increasing the tolerance of microbial cells to anthranilic acid and / or salts (oAB), the method comprising the following steps: The microbial cells are cultured in the presence of at least 50 g / l oAB and a fermentable substrate, alkali metal ions are added to the culture medium so that the molar ratio of the alkali metal ions to the oAB is in the range of 0.75 to 1.25, wherein the molar concentration of ammonium ions in the culture medium does not exceed 200 mM, wherein the microbial cells are bacterial cells belonging to the genus Corynebacterium, and wherein the alkali metal ions are sodium and / or potassium ions.

2. The method according to claim 1, wherein the microbial biomass reaches at least 6 g / l dry weight.

3. Use of alkali metal ions for increasing the tolerance of microbial cells to oAB at a concentration of at least 50 g / l, wherein the microbial cells are bacteria belonging to the genus Corynebacterium, wherein the alkali metal is sodium and / or potassium.

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