Methods for increasing the tolerance of microbial cells to anthranilic acid by limiting the amount of ammonia in the culture medium.

Abstract

The present invention relates to methods for increasing the concentration of anthranilic acid tolerated by a given microbial cell by limiting the concentration of ammonia in the culture medium.

Description

1 / 17 Descriptive Report of the Invention Patent for "METHODS FOR INCREASING THE TOLERANCE OF MICROBIAL CELLS TO ANTHRANILIC ACID BY LIMITING THE AMOUNT OF AMMONIA IN THE CULTURE MEDIUM"

[001] The present invention relates to methods for increasing the concentration of anthranilic acid tolerated by a given microbial cell by limiting the concentration of ammonia in the culture medium.

[002] Currently, there is no commercially available renewable or biologically derived source of oaminobenzoate or the corresponding acid. Current methods of aniline production rely on the chemical synthesis of petroleum-derived feedstocks. Such petroleum-derived feedstocks are non-renewable, as opposed to renewable feedstocks such as the renewable resource biomass. The chemical synthesis of aniline is a multi-step process. The various reaction steps involved in aniline production result in high production costs. Furthermore, conventional, i.e., chemical, synthesis of aniline is associated with hazardous intermediates, solvents, and waste products that can have substantial environmental impacts. Non-specific side reactions in the aromatic ring result in reduced product yield, thus further increasing production costs.Raw materials derived from petroleum are influenced by cost fluctuations resulting from the global price of oil.

[003] o-Aminobenzoate is a natural intermediate of the shikimate pathway and a precursor to the biosynthesis of the aromatic amino acid L-tryptophan. Document number WO 2015 / 124687 discloses a concept for the production of biologically derived aniline in two process steps: (1) the fermentative production of o-aminobenzoate using recombinant bacteria and (2) the subsequent catalytic conversion of o-aminobenzoic acid to aniline. The recombinant bacteria used in said process belong to the Corynebacterium or Pseudomonas family. Both bacteria produce o-aminobenzoate at pH values ​​between 7 and 8.

[004] The following problem exists when producing o-aminobenzoate at a pH in the range between 7 and 8: Due to the fermentative production of o-aminobenzoate, which is an acid, it is Petition 870200046331, dated April 13, 2020, pp. 64-87 2 / 17 It is necessary to add a base such as NH4OH to ensure a stable neutral pH. In this way, a salt of, for example, NH4+ / o-aminobenzoate is produced. However, these o-aminobenzoate salts are toxic to microbial cells. According to Figure 3, the metabolic activity of bacterial cells (see OTR) is limited when NH4+ / o-aminobenzoate concentrations above 25 g / l of o-aminobenzoate (not including the mass of the cation) are reached, and cell growth (see dry weight) stops at higher concentrations (>50 g / l). This toxicity is not known for products such as glutamate or lysine.

[005] This type of toxicity problem is usually solved by the direct evolution of the applied microbial cells. First, the microbial cells are exposed to increasing concentrations of the toxic component (e.g., oaminobenzoate) in repeated batch experiments or continuous fermentation assays. Thus, the microbial cells evolve by random mutagenesis (which can be accelerated by the addition of mutagens) and the most resistant microbial cells survive. Second, the most resistant cells are isolated / selected and can be used for production.

[006] However, many of the mechanisms underlying resistance in these microbial cells consume energy (Aindrila Mukhopadhyay. Trends in Microbiology, August 2015, Volume 23, Number 8; Rau et al. Microb Cell Fact (2016) 15: 176; Warnecke T, Gill RT. Microbial Cell Factories. 2005; 4: 25). Thus, a certain proportion of the fermentable substrate is consumed for maintenance metabolism, leading to reduced yields of o-aminobenzoate. For this reason, the high titers of o-aminobenzoate, necessary for a reasonable time-space yield of the process, concomitantly decrease the product yield.

[007] Although the biotechnological production of o-aminobenzoate from renewable sources as a precursor for aniline production offers potential benefits, the toxicity factor described above diminishes the potential benefits of this process. Therefore, alternative methods are needed to increase the resistance of microbial cells to o-aminobenzoic acid. Petition 870200046331, dated April 13, 2020, pp. 65-87 3 / 17

[008] This problem is solved by the methods defined in the claims and description below.

[009] In a first embodiment, the present invention relates to a method for cultivating at least one microbial cell capable of converting a fermentable substrate into oAB in the presence of the fermentable substrate while maintaining its metabolic activity, wherein the culture medium is characterized by an ammonia concentration not exceeding 200 mM and an ortho-aminobenzoic acid (oAB) concentration of at least 40 g / l.

[010] Preferably, the concentration of oAB in this modality is 40 g / lag / l, more preferably 40 g / lag 70 g / l, more preferably 40 g / lag 60 g / l.

[011] In this mode, metabolic activity is preferentially oxygen consumption, as determined by the oxygen transfer rate (OTR) of the culture. The culture is the suspension consisting of at least one microbial cell and the culture medium. Sustained metabolic activity of the cell or cells is indicated by a culture OTR that does not decrease. Preferably, sustained metabolic activity is an increasing culture OTR.

[012] Under the culture conditions set forth above, the so-called microbial cells convert at least a proportion of the fermentable substrate into oAB. Therefore, in another embodiment, the present invention relates to a method for producing ortho-aminobenzoic acid (oAB) by cultivating a microbial cell capable of converting a fermentable substrate into oAB in the presence of the fermentable substrate and under conditions suitable for the conversion of the fermentable substrate into oAB, wherein the ammonia concentration in the culture medium does not exceed 200 mM and the oAB concentration is at least 40 g / l.

[013] Preferably, the concentration of oAB in this modality is 40 g / lag / l, more preferably 40 g / lag 70 g / l, more preferably 40 g / lag 60 g / l.

[014] The microbial cell is preferably a cell that is capable of converting Petition 870200046331, dated April 13, 2020, pp. 66-87 4 / 17 Biologically, a fermentable substrate is converted into oAB. The term biological conversion refers to the biochemical processes that transform one or more molecules of the fermentable substrate into one or more molecules of oAB. These processes are predominantly mediated by enzymes expressed by the bacterial cell.

[015] The term o-aminobenzoic acid (or oAB), as stated in this application, refers to 2-aminobenzoic acid. This compound is also known as anthranilic acid. A person skilled in the art knows that an acid can be present in its protonated form as a neutral substance or deprotonated as an anion. In aqueous solution, a portion of the acid is protonated and a portion is present as an anion. The ratio of protonated acid to anion depends on the pH of the solution and the dissociation constant Ka of the acid in question. Unless otherwise indicated, the term o-aminobenzoic acid, as used in this application, always refers to the protonated acid and the corresponding anion.

[016] The microbial cell used in the present invention may be a naturally occurring strain, that is, a microbial strain that has no further human interaction, particularly without genetic manipulation, capable of converting a fermentable substrate into oAB. However, in a preferred embodiment of the present invention, it is a microbial cell that has gained the aforementioned capability in the process of genetic manipulation or a microbial cell in which such methods have been used to enhance a pre-existing capability.

[017] The term genetic modification within the meaning of the invention refers to alterations in the nucleic acid sequence of a particular gene of a microbial host compared to the wild-type sequence. This genetic modification may comprise deletions as well as insertions of one or more deoxyribonucleic acids. This genetic modification may comprise partial or complete deletions as well as insertions introduced by transformations in the genome of a microbial host. Such genetic modification may produce a recombinant microbial host, wherein said genetic modification may comprise alterations of at least one, two, three, four or more nucleotides. Petition 870200046331, dated April 13, 2020, pages 67 / 87 5 / 17 unique compared to the wild-type sequence of the respective microbial host. For example, a genetic modification may be a deletion or insertion of at least one, two, three, four or more unique nucleotides or a transformation of at least one, two, three, four or more unique nucleotides. A genetic modification according to the invention may have the effect of, for example, reduced expression of the respective gene or, for example, enhanced expression of the respective gene.

[018] The microbial cell is either a prokaryotic cell or a eukaryotic cell. Preferably, the prokaryotic cell is a bacterial cell. The preferred bacterial cells belong to the genera Corynebacterium, Mycobacterium, Bacillus, Pseudomonas, Escherichia, and Vibrio. Most preferred are Corynebacterium glutamicum and Pseudomonas putida. The most preferred is Corynebacterium glutamicum ATCC 13032. The preferred eukaryotic cells belong to the order Saccharomycetales or the genus Aspergillus. Most preferably, they belong to the species Ashbya gossypii, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Zygosaccharomyces bailii, Kluyveromyces marxianus, and Saccharomyces cerevisiae. With the highest preference, the yeast is Saccharomyces cerevisiae.

[019] It is particularly preferred that said microbial cell be characterized by a genetic modification of the trpD gene that prevents or reduces the expression of said gene and / or that leads to a gene product with reduced or no enzymatic activity. The expert in the technique can easily generate these microbial cells using conventional genetic methods.

[020] Recombinant bacterial cells that are particularly suitable for the method of the present invention are disclosed in document number WO 2015 / 124687.

[021] The term cultivar refers to the incubation of the microbial cell under conditions that facilitate metabolic activity. Such conditions are known to those skilled in the art. These conditions minimally encompass the presence of the microbial cell in a medium suitable for cell growth at temperatures that Petition 870200046331, dated April 13, 2020, pages 68 / 87 6 / 17 allow for cell proliferation, the presence of a fermentable substrate, and the presence of oxygen. Preferably, said metabolic activity is oxygen consumption. More preferably, metabolic activity is measured as one or more parameters selected from the group consisting of oxygen consumption, glucose consumption, growth rate, and OAB production. With the highest preference, metabolic activity is the biological conversion of the fermentable substrate into oAB or the growth rate.

[022] Preferably, the culture is carried out in a culture medium with a pH between 6.0 and 8.0.

[023] A fermentable substrate as understood in the present patent application is any organic compound or mixture of organic compounds that can be used by the microbial cell to produce o-aminobenzoic acid in the presence or absence of oxygen. Preferred fermentable substrates additionally serve as sources of energy and carbon for the growth of the microbial cell. Preferred fermentable substrates are processed beet, sugarcane, plants containing starch and lignocellulose. Also preferred as fermentable substrates are glycerol and C1 compounds, preferably CO, and fermentable sugars. A preferred fermentable sugar is glucose.

[024] The term culture medium is generally understood in the art. This refers to an aqueous solution that provides conditions that allow the metabolic activity of the microbial cell. These conditions are physical or physicochemical, such as temperature, dissolved oxygen concentration, ion strength, and pH. They are also chemical and include the concentration of the different nutrients required by the microbial cell for its activity. The person skilled in the art can adapt these conditions to the needs of a specific microbial cell based on the common knowledge available for that specific microbial cell.

[025] The term ammonia refers to ammonia in its neutral form, that is, NH3, as well as ammonium, that is, NH4+. “Ammonia concentrations”, therefore, always refer to the sum of the concentrations of both species. Petition 870200046331, dated April 13, 2020, pp. 69-87 7 / 17

[026] Aqueous ammonia is a popular buffer in biotechnological applications. In the production of organic acids, such as oAB, it can be used to maintain a stable pH despite increasing concentrations of the acid fermentation product. However, in the study underlying the present invention, it was surprisingly found that the absence of high ammonia concentrations due to the use of sodium hydroxide as a buffer substance leads to a sharp increase in the oAB concentration tolerated by the microorganism in question.

[027] Therefore, the ammonia concentration during oAB production by a microbial cell needs to be limited, to concomitantly limit the toxicity of the oAB product. The ammonia concentration present during oAB production does not exceed 300 mM, preferably 200 mM. More preferably, it reaches a maximum of 100 mM and most preferably 50 mM. It should be understood that the addition of aqueous ammonia or ammonium salts from stock solutions as a nitrogen source or as a component of a buffer system may lead to concentrations exceeding the aforementioned values ​​locally and for a limited period of time, because the uniform distribution of the added liquid in a fermentation vessel requires a certain amount of time. The same applies to the addition of ammonium salts in solid form, where the dissolution of the salt may lead to local peaks in ammonia concentration before the dissolved salt is diluted.

[028] Thus, the term “locally” refers to a proportion of no more than 10% of the total volume of the culture medium. Any peak in ammonia concentration in the culture medium should not last more than 15 minutes before mixing the culture medium leads to a distribution of the added compounds in the vessel, such that the above-defined limit levels are not exceeded.

[029] Since lower concentrations of ammonia do not cause excessive toxicity of oAB, in a preferred embodiment of the present invention, the culture medium contains 0.5 mM to 300 mM, preferably 0.5 mM to 200 mM, and most preferably 0.5 mM to 100 mM of ammonia. Preferably, the lower limit of the ammonia concentration is 10 mM. Thus, ammonia can still be used as part Petition 870200046331, dated April 13, 2020, pages 70-87 8 / 17 of the buffer system and can be used as a cheap and convenient nitrogen source.

[030] However, in another preferred embodiment of the present invention, ammonia is not actively added to the culture medium, so that the ammonia concentration does not exceed 5.0 mM, preferably 1.0 mM and more preferably 0.5 mM. In this embodiment, nitrogen sources other than ammonia need to be used. Alternative nitrogen sources are well known to those skilled in the art. Preferred alternative nitrogen sources are urea, amino acids, peptides, as well as complex mixtures comprising the aforementioned compounds. Mixtures comprising alternative nitrogen sources are preferably yeast extract, tryptone, casamino acids and corn steep liquor. Preferred amino acids are glutamine, threonine, alanine, asparagine and serine.

[031] In a preferred embodiment of the present invention, low concentrations of ammonia are supplemented with alternative sources of nitrogen. This is preferably done if the total ammonia concentration at any point in time during the crop cycle is in the range between 0.5 and 250 mM, more preferably 0.5 to 100 mM. A preferred alternative source of nitrogen to be used to supplement the aforementioned ammonia concentration is urea.

[032] Under the culture conditions defined above, OAB concentrations of at least 20 g / l are achieved, preferably at least 30 g / l and more preferably at least 40 g / l.

[033] Preferably, under the above-defined culture conditions, the microbial biomass reaches a biomass of at least 6 g / l, more preferably at least 9 g / l.

[034] In a particular preferred embodiment of the present invention, the microbial biomass reaches a biomass of at least 6 g / l, more preferably at least 9 g / l, under the culture conditions defined above and in the presence of at least 30 g / l oAB, more preferably at least 40 g / l oAB. Petition 870200046331, dated April 13, 2020, pp. 71-87 9 / 17

[035] Advantageously, the microbial cell maintains its metabolic activity up to concentrations of at least 30 g / l of oAB, more preferably at least 40 g / l of oAB and most preferably at least 50 g / l of oAB if the ammonia concentration in the culture medium does not exceed 300 mM, more preferably 250 mM and most preferably 100 mM. The cell maintains its metabolic activity if said metabolic activity is at least 50% of the activity in the absence of oAB. Preferably, metabolic activity is measured as one or more parameters selected from the group consisting of oxygen consumption, glucose consumption, growth rate and oAB production.

[036] It should be understood that any sudden increase in oAB concentration causes a transient decrease in the metabolic activity of the microbial cell (see Examples). During the oAB production process, this effect is unlikely to be found because oAB concentrations increase gradually, allowing the microbial cell time to adapt. However, the addition of oAB for testing purposes may have this effect. Therefore, it is preferable that the metabolic activity of a microbial cell be measured at least two hours after any sharp increase in oAB concentration, for example, caused by the addition of oAB to the culture medium. Otherwise, the true and lasting effect of oAB on activity may be overestimated for the given conditions.

[037] In a preferred embodiment of the present invention, aqueous ammonia is replaced by an alkali metal ion base as a buffering agent during microbial cell culture. Said alkali metal base is preferably sodium hydroxide.

[038] In another preferred embodiment of the present invention, no buffering agent is added during the microbial cell culture. A buffering agent may be omitted if the culture medium contains sufficient concentrations of CaCO3 from the beginning of the fermentation process or if urea is used as an alternative nitrogen source. Based on the amount of CaCO3 required to neutralize a given amount of oAB and the maximum acceptable drop in pH that is acceptable, Petition 870200046331, dated April 13, 2020, pp. 72-87 10 / 17 the initial concentration of oAB can be calculated. If the addition of a buffering agent during fermentation is omitted, 10 to 30 g / l, more preferably 15 to 25 g / l of CaCO3, are added to the culture medium before the start of fermentation.

[039] In another preferred embodiment, the present invention relates to the use of a culture medium with an ammonia concentration not exceeding 300 mM, preferably not exceeding 200 mM, for the biological conversion of a fermentable carbon source into oAB by a microbial cell capable of said conversion.

[040] All definitions given above for the method of the invention also apply to this embodiment, unless otherwise specified.

[041] More preferably, the ammonia concentration in the culture medium does not exceed 200 mM. Even more preferably, it does not exceed 100 mM. Most preferably, it does not exceed 5 mM.

[042] In yet another embodiment, the present invention relates to the use of a culture medium with an ammonia concentration not exceeding 10 mM, to increase the tolerance of microbial cells to oAB.

[043] All definitions given above for the method of the invention also apply to this embodiment, unless otherwise specified.

[044] The term use of a culture medium to increase the tolerance of microbial cells to OAB”

[045] The following examples are intended to illustrate the invention only. They shall not limit the scope of the claims in any way. EXAMPLES

[046] The Figures show: Figure 1: Evaluation of the maximum tolerable concentration of oAB during batch culture in a 1 L laboratory-scale bioreactor with a modified production strain of C. glutamicum. The addition of 20 mL of a 500 g / L oAB stock solution (oAB dissolved with NaOH at pH 7) after 23 h, 39 h, 48 h (twice), 64 h (twice), and 71 h (twice), and the addition of glucose solution after... Petition 870200046331, dated April 13, 2020, pp. 73-87 11 / 17 h, 48 h, 63 h, 65 and 71.5 h to avoid glucose limitation. Initial culture volume VL = 1 l, temperature 30 °C, pH = 7 controlled by the addition of NH4OH (10% by weight of NH3) during culture, gas emission rate with air = 0.2 l / min, pO2 controlled at 30% air saturation, adjusting the agitator speed between 200 and 1,200 rpm. Figure 2: Influence of Na-oAB and NH4-oAB addition on oAB resistance during 1 L scale batch culture with C. glutamicum ATCC 13032. Filled symbols: addition of 40 mL of 500 g / L oAB stock solution (oAB dissolved with NaOH at pH 7) after 7 h and 24 h. Open symbols: addition of 40 mL of 500 g / L oAB stock solution (oAB dissolved with NH4OH at pH 7) after 7 h and 24 h. Both bioreactors: addition of 36 g / L glucose (added as stock solution) after 7.5 h and 24.5 h to avoid glucose limitation. Initial culture volume VL = 1 l, temperature 30 °C, pH = 7 controlled with NH4OH (10% by weight of NH3), gas emission rate with air = 0.2 l / min, pO2 controlled with 30% air saturation, adjusting the agitator speed between 200 and 1,200 rpm. Figure 3: Influence of the addition of Na-oAB and NH4-oAB on the resistance of oAB during batch culture on a 1 l scale with C. glutamicum ATCC 13032. Filled symbols: addition of 40 ml of 500 g / l oAB stock solution (oAB dissolved with NaOH at pH 7) after 6.4 h and 23.6 h. Open symbols: addition of 40 ml of 500 g / l oAB stock solution (oAB dissolved with NH4OH at pH 7) after 6.4 h and 23.6 h. Initial culture volume VL = 1 l, temperature 30 °C, pH = 7 controlled with NH4OH (10% by weight of NH3), gas emission rate with air = 0.2 l / min, pO2 controlled with 30% air saturation, adjusting the agitator speed between 200 and 1,200 rpm. Figure 4: Influence of NaCl and NH4Cl addition on oAB resistance during 1 L batch culture with a modified production strain of C. glutamicum. Addition of 20 mL of 500 g / L oAB stock solution (oAB dissolved with NaOH at pH 7) after 23 h, 39 h and 47 h (twice) and addition of glucose after 19 h, 37 h, 47 h and 71 h to prevent glucose limitation. Solid lines: Petition 870200046331, dated April 13, 2020, pp. 74-87 12 / 17 Reactor 1, addition of 70 ml of a 244 g / l NaCl stock solution after 64 h. Dashed lines: Reactor 2, addition of 70 ml of a 223 g / l NH4Cl stock solution after 64 h. Initial culture volume VL = 1 l, temperature 30 °C, pH = 7 controlled with aqueous NH4OH (10% by weight NH3), gas emission rate with air = 0.2 l / min, pO2 controlled at 30% air saturation, adjusting the stirrer speed between 200 rpm and 1200 rpm. EXAMPLE 1: EVALUATION OF MAXIMUM CONCENTRATION TOLERABLE OAB OF A MODIFIED PRODUCTION STRAIN OF C. GLUTAMICUM.

[047] The maximum tolerable concentration of oAB in the presence of sodium ions in the medium was tested with a modified production strain of C. glutamicum. This strain was derived from Corynebacterium glutamicum ATCC 13032 by evolutionary engineering, to increase its resistance against oAB. This strain was then genetically modified to make it capable of producing oAB and comprised the following modifications. The incorporated modifications have the effect of reduced expression of the trpD gene, which encodes anthranilate phosphoribosyltransferase, knocking out the ppc gene, which encodes PEP carboxylase, constitutive overexpression of the aroGD146Ne and trpEGS40F genes, heterologs of the E. coli genes, which encode DAHP synthase and feedback-resistant anthranilate synthase, respectively, and constitutive overexpression of the E. coli aroL gene, which encodes shikimate kinase.

[048] A bioreactor with a nominal volume of 1 l was filled with sterile culture medium, including 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 CaCC2 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 of FeSO4^7H2O, 1 mg / l of ZnSO4TH2O, 0.2 mg / l of CuSO4^5H2O and 0.02 mg / l of NICC6H2O.

[049] The pre-culture medium for culture 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 pre-culture was grown Petition 870200046331, dated April 13, 2020, pp. 75-87 13 / 17 in 300 ml shaker bottles with a liquid volume of 25 ml at a temperature of 28 °C and a shaking frequency of 180 rpm until OD600 > 20 is reached.

[050] The culture was carried out in a laboratory-scale bioreactor with an initial culture volume of 1 l. The temperature was controlled at 30 °C and the pH was maintained constant at pH = 7 by adding aqueous NH4OH solution (10% by weight NH3) during fermentation. The gas emission rate was set to 0.2 l / min of air and the dissolved oxygen tension was controlled at 30% air saturation by controlling the stirrer speed between 200 rpm and 1200 rpm. The culture results are shown in Figure 1. The oAB concentration was gradually increased by adding 20 ml of a 500 g / l oAB stock solution (oAB dissolved with 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 h, and 71.5 h to avoid glucose limitation. An increase in biomass concentration was observed even at an oAB concentration of 80 g / L, as shown in Figure 1.Increasing the oAB concentration from 80 g / l to 100 g / l after 71 h resulted in a decrease in metabolic activity (indicated by the decline in OTR signal) and no further increase in biomass was observed at that point. This experiment demonstrated that growth of the C. glutamicum production strain in the presence of 80 g / l oAB can be achieved by adding oAB as a sodium salt instead of an ammonium salt to the bioreactor, thus avoiding high ammonium concentrations. EXAMPLE 2: COMPARISON OF METABOLIC ACTIVITY AFTER THE ADDITION OF NH4-OAB AND NA-OAB DURING THE CULTURE OF C. GLUTAMICUM ATCC 13032

[051] A C. glutamicum strain derived from ATCC 13032 was used to compare metabolic activity after the addition of NH4-oAB and Na-oAB during culture. This strain was derived from Corynebacterium glutamicum ATCC 13032 through evolutionary engineering to increase its resistance to oAB. It was not further genetically modified. Therefore, it was not capable of producing oAB. For this purpose, Petition 870200046331, dated April 13, 2020, pages 76 / 87 14 / 17 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 / l KH2PO4, 1 g / l K2HPO4, 0.25 g / l MgSO4^7 H2O, 0.01 g / l CaCb2 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 FeSÜ4^7H2O, 1 mg / l ZnSO4^7H2O, 0.2 mg / l CuSO4^5H2O and 0.02 mg / l of NiCl2-6H2O.

[052] The pre-culture medium for culture 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 pre-culture was grown 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 OD600 > 20 was reached.

[053] The results for dry biomass, oAB and NH4 concentrations, and oxygen transfer rate (OTR) signals are shown in Figure 2. After adding 40 ml of a 500 g / l oAB stock solution (oAB dissolved with NaOH at pH 7) at a culture time of 7 h and 24 h in reactor 1, the biomass concentration and OTR signal continued to increase (filled symbols in Figure 2). In contrast, adding NH4-oAB (added by injecting 40 ml of a 500 g / l oAB stock solution containing oAB dissolved with NH4OH at pH 7) to reactor 2 after 24 h resulted in a constant OTR signal and reduced biomass accumulation (open symbols in Figure 2). This experiment shows that replacing ammonia with sodium as a counterion for oAB increases the tolerance of C. glutamicum ATCC 13032 to oAB. EXAMPLE 3: COMPARISON OF METABOLIC ACTIVITY AFTER THE ADDITION OF NH4-OAB AND NA-OAB DURING THE CULTURE OF C. GLUTAMICUM ATCC 13032

[054] The influence of ammonium was tested with C. glutamicum ATCC 13032 (without other genetic modifications, not modified by evolutionary engineering) using two 1 l bioreactors filled with sterile culture medium, including the following initial concentrations: 20 g / l glucose, 5 g / l (NH4)2SO4, 1 g / l KH2PO4, Petition 870200046331, dated April 13, 2020, pages 77 / 87 15 / 17 g / l K2HPO4,0.25 g / l MgSO4·7 H2O, 0.01 g / l CaCb2 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 of FeSO4·7H2O, 1 mg / l of ZnSO4·7H2O, 0.2 mg / l of CuSO4·5H2O and 0.02 mg / l of NiCl2·6H2O.

[055] The pre-culture medium for culture 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 pre-culture was grown 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 OD600 > 20 was reached.

[056] During fermentation, the temperature was controlled at 30 °C and the pH was kept constant at pH=7 by adding an aqueous solution of NH4OH (10% by weight of NH3). The gas emission rate was adjusted to 0.2 l / min of air and the dissolved oxygen tension was controlled at 30% air saturation by adjusting the agitator speed between 200 rpm and 1,200 rpm.

[057] The results of dry biomass and OAB concentrations and the signals related to the oxygen transfer rate (OTR) are shown in Figure 3. 40 ml of a 500 g / l oAB stock solution (oAB dissolved with NaOH at pH 7) was added to reactor 1 after 6.4 h and 23.6 h (filled symbols) and 40 ml of a 500 g / l oAB stock solution (oAB dissolved with NH4OH at pH 7) was added after 6.4 h and 23.6 h to reactor 2 (open symbols)

[058] As shown in Figure 3, biomass accumulation continued after the addition of Na-oAB to reactor 1 (filled symbols in Figure 3). In contrast, the addition of NH4-oAB to reactor 2 caused an inhibition of growth after 24 h (open symbols in Figure 3). It follows that the toxicity of oAB in C. glutamicum ATCC 13032 in the presence of high sodium concentrations is reduced compared to the toxicity in the presence of high ammonium concentrations. EXAMPLE 4: COMPARISON OF METABOLIC ACTIVITY AFTER THE ADDITION OF NACL AND NH4CL DURING THE CULTURE OF A STRAIN OF Petition 870200046331, dated April 13, 2020, pages 78 / 87 16 / 17 MODIFIED PRODUCTION OF C. GLUTAMICUM

[059] The strain was modified as explained in example 1. To evaluate whether an increase in tolerance to oAB is caused by the absence of high concentrations of ammonium or by the presence of sodium in the medium, the influence of the addition of NaCl and NH4Cl on the toxicity of oAB was tested. Two bioreactors with a nominal volume of 1 l were filled with sterile culture medium, including 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 CaCb2 H2O, 2 mg / l biotin (vitamin B7), 0.03 g / l protocatechuic acid (3,4-dihydroxybenzoic acid), 0.01 g / l MnSO4^O. 0.01 g / l FeSOW^O, 1 mg / l ZnSO4^7H2O, 0.2 mg / l CuSO4^5H2O and 0.02 mg / l NiCb6H2O.

[060] The pre-culture medium for culture 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 pre-culture was grown 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 OD600 > 20 was reached.

[061] The culture was carried out in laboratory-scale bioreactors with an initial culture volume of 1 l. The temperature was controlled at 30 °C and the pH value was maintained constant at pH 7 by adding aqueous NH4OH solution (10% by weight NH3) during fermentation. The gas emission rate was adjusted to 0.2 l / min of air and the dissolved oxygen tension was controlled at 30% air saturation by adjusting the stirrer speed between 200 rpm and 1200 rpm. Two bioreactors were inoculated with the production strain. The culture results are shown in Figure 4. The oAB concentration was gradually increased in both bioreactors by adding 20 ml of a 500 g / l oAB (NaoAB) stock solution after 23 h, 39 h and 47 h (twice). Glucose was added after 19 h, 37 h, 47 h, and 71 h to prevent glucose limitation.After a culture time of 64 h, a volume of 70 ml of water containing 244 g / l of NaCl was added to bioreactor 1 (solid lines in Figure 4), resulting in an additional quantity of 292 mmol. Petition 870200046331, dated April 13, 2020, pp. 79-87 17 / 17 of Na+ ions. An equimolar amount of NH4Cl was added to reactor 2 by adding 70 ml of a 223 g / l NH4Cl stock solution after 64 h (dashed lines). The inline signals for the oxygen transfer rates (OTR) for both bioreactors are shown in Figure 4. The addition of NaCl had no effect on the OTR signal of reactor 1. In contrast, the OTR signal of reactor 2 decreased by approximately 20% after the addition of the NH4Cl solution (Figure 4). The drop in metabolic activity affected the final dry weight concentration, which reached 7.8 g / l in reactor 1 and 6.8 g / l in reactor 2 after a culture time of 91 hours. The results demonstrated that increasing ammonium concentrations leads to reduced metabolic activity of the C. glutamicum production strain.

[062] The effects described above can be generalized at least to the genus Corynebacterium. A strain subjected to evolutionary engineering, a method that alters the genetic material of an organism in a random manner, shows the same behavior as the strain used in example 3. Thus, the effect seems to be based on some quite fundamental functions of the cell that are not easily altered. Petition 870200046331, dated April 13, 2020, pages 80 / 87

Claims

1 / 2 CLAIMS 1. A method for cultivating at least one cell of Corynebacterium glutamicum capable of converting a fermentable substrate into oAB in the presence of the fermentable substrate while maintaining its metabolic activity, wherein the culture medium is characterized by an ammonia concentration not exceeding 200 mM, a pH between 6.0 and 8.0, and an ortho-aminobenzoic acid (oAB) concentration of 40 g / l to 80 g / l.

2. A method according to claim 1, characterized in that the sustained metabolic activity is indicated by a rate of oxygen transfer from the culture that does not decrease.

3. Method according to claim 1, characterized in that the oAB concentration is 40 g / l 60 g / l and at least a proportion of the fermentable substrate is converted to oAB by at least one cell.

4. Method according to claim 2, characterized in that the ammonia concentration in the culture medium does not exceed 5 mM.

5. A method, according to any one of claims 3 to 4, characterized in that the culture medium contains an alternative nitrogen source other than ammonia.

6. Method according to claim 5, characterized in that the alternative nitrogen source is urea.

7. Method according to claim 5, characterized in that the nitrogen source is selected from the group consisting of glutamine, alanine, asparagine, serine, threonine, peptides, and complex mixtures containing nitrogen.

8. A method according to any one of claims 3 to 7, characterized by comprising an additional step of adding an alkaline base to the culture medium as a buffering agent.

9. Method, according to any one of claims 1 to 8, characterized in that microbial biomass reaches at least 6 g / l in dry weight.

10. Use of a culture medium characterized by having an ammonia concentration not exceeding 200 mM, a pH between 6.0 and 8.0, and an oAB concentration of 40 g / l to 60 g / l for the biological conversion of a fermentable carbon source into oAB by a Corynebacterium glutamicum cell capable of said conversion. (Petition 870260065872, dated 03 / 07 / 2026, page 12 / 14)

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