DD290212A5 - PROCESS FOR PREPARING A GLUCOSE MINIMUM MEDIUM FOR ESCHERICHIA COLI MASS CULTURE FERMENTATION - Google Patents

Abstract

The invention relates to a process for the preparation of a minimal glucose medium for Escherichia coli mass culture fermentation for obtaining recombined DNA products, which is characterized in that {minimal glucose medium; fed-batch cultivation; Mass culture fermentation; Escherichia coli cells; recombinant Escherichia coli cells; recombinant DNA products; dry cell; p H value control; low level}

Description

Field of application of the invention

The invention relates to a process for the preparation of a minimal glucose medium for fermentations of Escherichia coli cells, in particular of those carrying plasmids with genes which code for recombinant DNA products, up to very high concentrations of dry biomass. Field of application of the invention is the microbiological or pharmaceutical research and industry.

Characteristic of the known state of the art

Escherichia coli is used as a bacterial host for the production of various recombinant DNA products (eg hormones, antivirals, immunomodulators, enzymes, etc.). To obtain high levels of the desired recombinant DNA products, in addition to a suitable cellular expression system, it is necessary to cultivate the Escherichia coli culture to high levels of bio-dry matter (X). By appropriate cultivation methods, namely by fed-batch fermentations, X values of more than 40 g could be achieved with E. coli (PAN, J. G. RHEE, JS and LEBEAULT, JM, 1987, Biotechnol. Letters, vol.9, No.2,89-94; MORI, H., YANO, T., KOBAYASHI, T. and SHIMIZU, S., 1979, J. Chem. Engineering, vol.12, No.4,313-319; Fieschko ₁ J. and RITCH, T, 1986, Chem.Eng.Commun, vol.45,229-240;.. Shiloach, J., and Bauer, S., 1975, Biotechn and Bioeng, vol 17.227 to 239).... In these fed-batch fermentations, the cultivation of E. coli cells is usually carried out in so-called glucose mineral salt media, which are often supplemented with complex substrates, such as yeast extract or peptones. In the production of these glucose-mineral salt media, strong precipitations occur.

These precipitations of nutrient solution components are often the cause of step limits and make an optical growth measurement in on-line operation impossible as a prerequisite for targeted control methods. In addition, a number of substrate components are grossly oversized, as shown by an estimate of the yield coefficients known from the literature (IRIT, S.J. 1975, Principles of Microbial and Cell Cultivation, Blackwell Scientific Publ./Oxford- London- Edinburgh-Melbourne). Therefore, the conversion levels of such substrate components are low and the nutrient media designed overall not economically advantageous. While the o.g. Fed-batch fermentations, the pH of the culture solution by alkaline solutions such. B. by ammoniacal solution, which also serves as a nitrogen source, or maintained by NaOH. In the feeding phase, glucose is used primarily as a feeding substrate.

For all o. G. It is also necessary process, in the course of fermentation additionally several salts and z.T. also other substrates, e.g. Vitamins, amino acids or yeast extract, to dose. This is disadvantageous because additional dosage systems are necessary and in particular since a constant monitoring of the fermentation must be carried out with the appropriate dosages at the appropriate times.

Object of the invention

The invention aims to provide a minimum glucose-free medium in which Escherichia coli cells can be easily fermented to high concentrations of dry biomass.

Explanation of the essence of the invention

Dor invention has for its object to describe a method for producing a glucose-minimal medium in which Escherichia coli cells can be cultivated in a fed-batch process in a simple manner to very high concentrations of dry biomass without strong precipitates in the preparation of the nutrient solution occur, and without after the inoculation of the nutrient solution during fermentation steady doses or one or more additions of additional nutrient solution components are required.

According to the invention, this object is based on a fermentation batch of about 141 as follows: For an approach of this volume for a fed-batch process for the cultivation of Escherichia coli with conventional glucose dosage and pH control 117.2 g KH ₂ PO ₄ ,> 15 g of citric acid and 35 g (NH ₄ I ₂ HPO ₄ separately dissolved in 11 distilled water and then mixed with oil distilled water.Then 20 ml of iron citrate solution [2.7Gew .-%], and with

20mlSpurengemischlösung (0,2GeW ^_1-0 ZoCoCI ₂ · 6H ₂ O, 1,2Gew .-% MnCl ₂ .4H ₂ 0,0,113Gew .-% CuCl ₂ · H ₂ 0,0,25Gew .-% H ₃ BO ₃ , 0.2Gew .-% N ₂ MoO ₄ · 2 H _2, 0.5 wt .-% citric acid) was added and with distilled water. filled to a volume of 8.51. This solution is steam sterilized in the fermentor for 15 minutes to 60 minutes, preferably 30 minutes. After cooling, 1 liter of glucose solution [5% by weight to 75% by weight of glucose] and 20 ml of zinc acetate solution (0.63% by weight of Zn (CH ₃ COO) ₂ , 0.372% by weight of EDTA] and after adjustment of the aclicity with 25% NH ₃ to pH 6.9 11 magnesium sulfate solution [0.33 wt% MgSO ₄ .H ₂ O). Finally, the acidity is readjusted with 25% NH ₃ to pH 6.5 to pH 6.9, preferably pH 6.7.

The trace mixture solution is prepared by first ₂ g CoCI ₂ 6H ₂ 0.12g MnCI ₂ Ί H ₂ 0,1,13g CuCl ₂ H ₂ O in 50OmI Aqua dest. with the addition of ₃ g of citric acid by boiling in solution and parallel to 2.5 g H ₃ BO ₃ with the addition of 1 g of citric acid in 10OmI aqua dest. be solved by cooking. Furthermore, 2 g of Na ₂ MoO ₄ 2 H ₂ O are added in parallel with the addition of 1 g of citric acid in 10OmI aqua dest. boiled and these three parallel mixtures after cooling to room temperature and mixed with distilled water. filled to 11.

The iron citrate solution is prepared by adding 27 g of iron citrate In 11 distilled water. be solved.

The glucose solution is prepared by adding 75g to 900g of glucose in 11 distilled water. are dissolved and the resulting solution is then autoclaved / sterilized.

The zinc acetate solution is prepared by adding 6.3 g of Zn (CH ₃ COO) ₂ 2 H ₂ O with the addition of 3.72 g of EDTA in 100 ml of distilled water. dissolved by boiling, then with distilled water. made up to 11 and the resulting solution is filtered sterile. The magnesium sulfate solution is prepared by adding 33 g MgSO ₄ .H ₂ O in 11 distilled water. dissolved and the resulting solution is then sterilized.

In addition, auxotrophic marker substances and selection marker substances according to the auxotrophies and selection markers of the strains to be fermented in this medium are added to the glucose minimal medium. To avoid foaming during the fermentation, anti-foaming agents are added to the batch of the glucose mini-medium in a manner known per se.

For a preferred embodiment of the invention, the auxotrophic marker substance thiamine, when inoculated with Escherichia coli strains carrying the genes with resistance-encoded genes, additionally carry the markers corresponding to the resistance to the minimal glucose medium when inoculated with the Escherichia coli strains TG1 or SK1590 , such as ampicillin, tetracycline or erythromycin and when inoculated with the recombinant E. coli strains, such as TG 1 / pBB210 or SK1590 / pHRW400, thiamine and ampicillin.

In the described approach fermentations up to dry matter concentrations of 70g Γ 'are possible. When using the glucose minimum medium for fermentations up to dry matter concentrations of 70g Γ ¹ to 20g Γ ¹ , the proportions of the substrates according to the specified production method for the minimal glucose medium are to be used as a percentage of the stated values for dry matter concentrations of 70g Γ 'reduced. This avoids the commonly occurring precipitate formations which occur when nutrient media in which dry biomass concentrations are to be produced above about 20 g.sup.- ¹ are used.

After inoculation in the fed-batch phase, glucose is added to the glucose minimal medium in a concentration of 700 g.sup.- ¹ . To maintain the pH, ammoniacal solution is added to the minimal glucose medium after being mopped.

In the preparation of the glucose mini-medium for other Ansa'zvolumina the individual nutrient solution components are to be changed in percentage. In this way, it is particularly advantageous to produce, in particular, minimal glucose media up to batch volumes of 500 l

 The glucose-minimal medium used according to the described preparation process has one before inoculation

Extinction value of E f ^{70 nm} -1.1 (measured in a 1 cm cuvette at a wavelength of 450 nm). This compared to 1cm

Distilled water (E 470nm "n, 02) measured under appropriate conditions, slightly higher value is due to a slight 1cm

causing precipitate-induced turbidity. After reaching a dry biomass concentration of about 15 g · Γ, measurements of the extinction, from which the dry biomass concentrations can be calculated using a calibration curve, are determined to be too large by only about 3% due to precipitation. This small error, especially when incorporating a correction factor, allows on-line optical measurement and calculation of dry biomass concentration as the basis for targeted fermentation control.

In the following, the procedure for the preparation of the minimal glucose medium will now be described in more detail, wherein the quantitative data refer to the approach of 141:

117.2 g KH ₂ PO ₄ ,> 15 g citric acid and 35 g (NH ₄ I ₂ HPO ₄ are individually dissolved in 11 distilled water, mixed with 51 distilled water and after addition of 20 ml iron citrate solution (27 g · Γ ¹ ) and 20 ml track mixture solution (preparation:.. First, ₂ 6H ₂ 0.12 g of MnCl ₂ .4H ₂ 0,1,13g CuCl ₂ · H ₂ O with the addition of 3 g of citric acid brought by boiling in 500ml of distilled water 2g CoCI solution in parallel, 2.5 g of H ₃ BO _{3 are} dissolved by boiling with addition of 1 g of citric acid in 100 ml of distilled water.Also, ₂ g of Na ₂ MoO ₄ 2 H ₂ O are boiled in 100 ml of distilled water with the addition of 1 g of citric acid are cooled to room temperature and give, filled with distilled water to 11, the trace mixture solution.) With distilled water to 8.51 This solution is then with steam at a temperature of 121 ⁰ C, an overpressure of 105 sterilized over a period of 30 minutes, the volume of A Through the formation of condensate during steam sterilization, the rate rises to 111 to 131. After cooling the heat-sterilized solution to a temperature below 50 ⁰ C successively solutions are followed: 11 autoclaved glucose solution (75g to 900g glucose in 11 distilled water.) And 20ml Zinkacetatlösu ^. (Preparation: 6.3g Zn (CH ₃ COO ₂ × 2H ₂ O are distilled in 100 ml of distilled water with addition of 3.72 g EDTA, then made up to a volume of 11 with distilled water and sterile filtered). Before the pH of the nutrient solution thus prepared is adjusted to pH 6.9 with 25% strength ammoniacal solution, the addition of sterile solutions with auxotrophic marker and selection marker is still necessary if necessary. Later, the glucose-minimal merlium z. B. inoculated with the E. coli strain TG1 or SK1590, then added as an auxotrophic marker thiamine, namely 40 mg in aqueous solution. If the E. coli strains obtain plasmids with ampicillin resistance encoding genes, such as pBB210 or pHRW400, then ampicillin is also added in aqueous solution. After adjusting the pH value until then

prepared nutrient solution to pH 6.9, the addition of 11 magnesium sulfate solution (33 g MgSO ₄ H ₂ O in 11 Aquadest, autoclave! art). As a result, the pH drops. The nutrient solution is then adjusted again in the fermenter to pH 6.8 with 25% ammoniacal solution. The thus prepared glucose minimum medium of about 141 is thus ready for the inoculation with E. coli.

The glucose minimal medium thus has the following quantitative composition:

KH ₂ PO ₄ (7.8 g · Γ ¹ ), (NH ₄ ) ₂ HPO ₄ (2.3 g · Γ '), MgSO ₄ · H ₂ O (2.2 g · Γ'), citric acid (> 1.0 g Iron citrate (36 mg Γ "), CoCl, 6 6H ₂ O (2.7 mg · Γ ¹ ), MnCl ₂ · 4H ₂ O (16 mg · Γ ¹ ), CuCl ₂ · H ₂ OH, 5 mg · l ^"1), H ₃ BO ₃ (3.3 mg · Γ ^1), Na ₂ MoO ₄ · 2H ₂ O (2.7 mg · I" ^1), (Zn (CH ₃ COO) ₂ · 2H ₂ O 8, 4 mg · Γ ^1), EDTA (5 mg · I ^"1), glucose (5 g · I" ¹ to 60 g · I "') and (as needed, for example, 2.7 mg thiamine Γ ¹⁾ and ampicillin (50 to 100 mg · Γ ¹ ) or other auxotrophic markers or selection markers.

The procedurally prepared glucose-minimal medium is characterized in addition to its low precipitate formation in particular by the fact that after inoculation substrates used by the growth of E. coli during the fed-batch fermentation with dosage of glucose (70Og Γ ¹ ) and pH control be almost completely metabolized with 25% ammoniacal solution to high cell densities (X »70g · Γ ¹ ).

The advantages of the present invention over the hitherto known technical solutions are summarized in particular in that

1. The produced glucose-minimal medium already contains all nutrient salts for fed-batch fermentations with E. coli up to high dry matter concentrations (X »70 g · Γ '), without once again having to carry out salt dosing in the fed-batch phase, so that additional contamination risks are eliminated.

2. the individual substrates in the glucose-minimal medium, the glucose fed in the fed-batch phase and the amount of NH ₃ introduced for pH maintenance are almost completely metabolized, and

3. The minimal glucose medium has only a slight precision-caused turbidity due to the special preparation procedure.

embodiment

In the following, the preparation of the minimal glucose medium is described, in the E. coli TG 1 / pBB210 for the recovery of recombinant human interferon alpha 1 up to high dry matter concentrations (X = 70g Γ ¹ ) by a fed-batch method with dosage of glucose and pH control with 25% ammonia, without the need for further additions of nutrient solution components during fermentation. Manufacturing regime:

The following components are separated into 11 distilled water. dissolved and destilled to 5l aqua. added: KH ₂ PO ₄ (117.2 g), citric acid (15 g), (NH ₄ ) ₂ HPO ₄ (35 g). After addition of 20 ml of iron citrate solution (27 g of iron citrate per 11 distilled water) and 20 ml of trace mixture solution, the filling with distilled water. to 8.51. This nutrient solution is steam sterilized in a stirred tank fermentor of the Chemap 30 type (time: approx. 30 min, temperature: 121 ° C, overpressure: approx. 105 kPa). Condensation during sterilization increases the volume of the reaction to 111 to 131.

The trace mixture solution is prepared as follows: First, in 500 ml of distilled water. 2g CoCI ₂ 6H ₂ 0.12 g of MnCl ₂ .4H ₂ 0,1,13g CuCl ₂ · H ₂ O made with the addition of 3 g of citric acid by boiling in solution. In parallel, 2.5 g of H ₃ BO ₃ with the addition of 1 g of citric acid in 100 ml of distilled water. solved by cooking. Furthermore, ₂ g of Na ₂ MoO ₄ 2 H ₂ O are added in parallel with the addition of 1 g of citric acid in 100 ml of distilled water. cooked. These three parallel mixtures are mixed after cooling to room temperature and distilled to 11 with distilled water. refilled. Dio solution thus prepared is the trace mixture solution, of which 20 ml are used.

To the steam-sterilized broth in Chemap 30, after cooling, the following individually prepared components are added under stirring in the following order:

First, 1.11 glucose-thiamine solution, secondly, 20 ml of zinc acetate solution, and third, 60 ml of the selection marker ampicillin. The three solutions are prepared separately as follows: Glucose-thiamine solution:

375 glucose are distilled into 11 distilled water. autoclaved. In parallel .n another vessel are 40mg of thiamine distilled in 100ml of aqua. were solved, also autoclaved. After cooling, the thiamine solution is introduced into the glucose solution. Zinkacetattösung:

6.3 g of Zn (CH ₃ COO) ₂ - 2 H ₂ O are dissolved in 10 liters of distilled water with the addition of 3.72 g of EDTA. cooked, then to 11 with distilled water. filled in and filtered sterile

 ampicillin:

1.5 g of ampicillin are distilled in about 60 ml of distilled water. dissolved and filtered sterile.

After addition of the above three solutions for steam-sterilized approach, the pH is 25% igerr. Ammonia adjusted to pH 6.9. Then 11 magnesium sulfate solution (33 g MgSO ₄ H ₂ O in 11 distilled water, autoclaved) is added, which is associated with a decrease in the pH. The pH is now adjusted to pH 6.8. Then 250 ml of seed suspension E. coli TG1 'pBB 210 are added and the fermentation started. The culture volume at the beginning of the fermentation is thus 131 to 151. The inoculated minimal glucose medium has the following composition at a volume of 151: KH ₂ PO ₄ (7.8 g × ¹ ). ((NH ₄ J ₂ HPO ₄ (2.3 g · Γ ^1), MgSO ₄ · H ₂ O (2.2 g · Γ ^1), citric acid (ca. 1.0 g I ^"1), ferric citrate 36mg · Γ ¹⁾ , CoCl ₂ · 6H ₂ O (2.7 mg · Γ ¹ ), MnCl ₂ · 4H ₂ O (16 mg · l ^-1 ), CuCl ₂ · H ₂ O (1.5 mg · l ^-1 ), H ₃ BO ₃ (3,3mg Γ ¹ ), Na ₂ MoO ₄ .2H ₂ O (2,7mg. Γ '), Zn (CH ₃ COO) ₂ - 2H ₂ O (8,4mg. R'), EDTA (5mg ¹ ), glucose (25g l "') and ampicillin (100mg · Γ'). During the course of the fermentation, the initial volume increases by the controlled dosage of 25% NH ₃ to maintain the pH and through the glucose dosage Final volume of the culture solution, with the high dry matter concentration of X "70g!", Is about 161 to 2Ol.

Claims (14)

1. A method for producing a glucose-minimal medium for Escherichia coli Massenkulturfermentation up to high dry matter concentrations by a fed-batch process with conventional glucose dosage and pH control, characterized in that for an approach of about 141 117.2 g KH ₂ PO ₄ , 15 g citric acid and 35 g (NH ₄ I ₂ HPO ₄ , separately dissolved in 11 distilled water each, then mixed with 51 distilled water, with 20 ml iron citrate solution [2.7 wt. "As well as with 20ml track mixture solution '0,2Gew .-% CoCI ₂ 6H ₂ 0,1,2Gew .-% MnCl ₂ .4H ₂ O, 0,113Gew .-% CuCl ₂ · H ₂ 0,0,25Gew .-% F ₃ BOa, 0.2 wt.% N ₂ MoO ₄ .2H ₂ .0.5 wt.% Citric acid) and made up to a volume of 8.51 with aqua-det. - Thereafter, this solution in the fermentor for 15 minutes to 60 minutes, preferably 30 minutes, steam sterilized, - After cooling 11 glucose solution [5 to 75Gew .-% glucose] and 2OmI zinc acetate solution [0.63Gew .-% Zn (CH ₃ COO) ₂ , 0.372 wt .-% EDTAl and after adjusting the pH with 25% NH _{3 is added} to pH 6.9 ¹ L of magnesium sulfate solution [0.33 wt% MgSO ₄ .H ₂ O] and - Shooting resetting the pH with 25% NH ₃ to pH 6.5 to pH 6.9, preferably pH 6.8 made 2. The method according to claim 1, characterized in that the trace mixture solution is prepared by - first 2gCoCI ₂ -6H ₂ 0,12g MnCl ₂ -4H ₂ OJ ₁ ISg CuCl ₂ - H ₂ O in 500ml aqua dest. with the addition of 3 g of citric acid brought into solution by boiling, - parallel to this 2.5g H ₃ BO ₃ with the addition of 1g citric acid in 10OmI aqua dest. solved by cooking, - In parallel, continue 2 g of Na ₂ MoO ₄ - 2 H ₂ O with the addition of 1 g of citric acid in 10OmI aqua dest. cooked and mixed these three parallel mixtures after cooling and with distilled water. be filled to 11. 3. proceed. according to claim 1, characterized in that the iron citrate solution is prepared by adding 27 g iron citrate in 11 distilled water. be solved. 4. The method according to claim 1, characterized in that the glucose solution is prepared by distilled 75g to 900g of glucose in 11 distilled water. are dissolved and the resulting solution is then sterilized. 5. The method according to claim 1, characterized in that the zinc acetate solution is prepared by 6,3g Zn (CH ₃ COO) ₂ · 2H ₂ O with the addition of 3.72 g EDTA in 10OmI aqua dest. dissolved by boiling, then with distilled water. made up to 11 and the resulting solution is filtered sterile. 6. The method according to claim 1, characterized in that the magnesium sulfate solution is prepared by 33 g MgSO ₄ · H ₂ O in 11 distilled water. dissolved and sterilizes the resulting solution ansxhließend. 7. The method according to claim 1 to 6, characterized in that the glucose-minimal medium in addition auxotrophic marker substances and selection marker substances are added in accordance with the auxotrophies and selection markers to be fermented in this medium strains. 8. The method according to claim 7, characterized in that the glucose-minimal medium when inoculated with the Escherichia coli strains TG1 or SK1590 additionally the auxotrophic marker substance thiamine is added. 9. The method according to claim 7 and 8, characterized in that the glucose-minimal medium when inoculation with Escherichia coü strains carrying plasmids with resistance-encoded genes, the resistance corresponding markers such as ampicillin, tetracycline or erythromycin are added. 10. The method according to claim 7 to 9, characterized in that the glucose-minimal medium when inoculated with the recombinant strains E. coli TG 1 / pBB210 or SK1590 / pHRW400 thiamine and ampicillin are added. 11. The method according to claim 1 to 6, characterized in that the glucose-minimal medium after inoculation in the fed-batch phase, glucose in a concentration of 700g · Γ ^{1 is} metered. 12. The method according to claim 1 to 6, characterized in that the glucose-minimal medium after inoculation to maintain the pH ammoniacal solution is added. 13. The method according to claim 1 to 6, characterized in that the production of the minimal glucose medium for any batch volumes, the individual nutrient solution components are changed percentage. 14. The method according to claim 1 to 6, characterized in that when using the glucose-minimal medium for fermentations up to Biotrockenmassekonzentrationen of 70g · I " ¹ to 20g · Γ ^1, the proportions of the substrates in the glucose-minimal medium in accordance with the percentage of the values for Dry biomass concentrations of 70g · Γ ¹ can be reduced.