DD299380A7 - METHOD FOR THE HIGH CELL DENSITY FERMENTATION OF ESCHERICHIA COLI IN A RUFFLER RESERVOIR - Google Patents

Abstract

The invention relates to a microbiological process for the fermentation of Escherichia coli strains, including recombinant E. coli strains, to high concentrations of dry biomass. It pursues the goal of achieving these synthesis achievements in stirred tank fermentors with as much as possible substrate utilization. The object of this aim is achieved by subjecting the fermentation of the included E. coli culture under aerobic conditions in a medium containing nitrogen source, organic carbon source and mineral salts, preferably in a glucose-mineral salt medium of well-defined composition, in sections, wherein first a batch section with maximum specific growth rate and then a Fed-batch section with submaximal specific growth rate is provided. The respectively desired submaximal specific growth rate is set here by variation of the oxygen input and monitored by controlling the oxygen content of the fermentor exhaust air. According to the proposed method, E. coli cultures with great stability can be fermented to dry biomass concentrations in the high cell density range {microbiological process; Escherichia coli; High cell density fermentation; Ruehrkesselfermentor; Nutrient medium with organic carbon source; Glucose-mineral salts medium; partial fermentation; Oxygen content of fermenter exhaust air}

Description

Field of application of the invention

Oils invention relates to a method for high cell density fermentation of E. coli strains, including those useful as hosts for vectors with recombinant expression systems.

The field of application of the invention is generally in those industries of an economy which comprises a biotechnology sector. jlsen and use in this E. coli strains as producer orphan.

Characteristic of the known state of the art

E. coli is often used as a cellular host for the production of recombinant DNA products. In addition to a high intracellular concentration of the desired product, the achievement of high levels of cells in the fermentor is a crucial prerequisite for high overall yield.

When cultivating to high cell densities in the conventional stirred tank fermenter, the problems of growth inhibition are confronted by too high substrate initial concentrations, the consumption of essential nutrient media components in the course of cultivation, the formation of inhibitory by-products of the metabolism and the limited capacity of oxygen supply.

The increasing demand for oxygen from a growing E.coli culture can be overcome by several strategies, namely: increase in stirrer speed and gassing rate, supply of oxygen-enriched air (Jung et al., 1988; Fass et al., 1989; Krüger 1989; 1989), Bauer and Ziv 1976, Mori et al., 1979, Gleiser and Bauer, 1981, Mitzutani et al., 1986, Baily et al., 1989), fumigation with pure oxygen (Bauer and Shiloach 1974; 1987, Pan et al 1987, Krüger 1989), low temperature cultivation (Shiloach and Bauer 1975, Bauer and White 1976, Bauer and Ziv 1976), and elevated pressure (Tsai et al., 1987).

With excess oxygen, E. coli was fermented in a glucose-mineral salt medium up to 16.5 g of dry biomass X / l (Reiling et al., 1985). Krüger (1989) and Eppstein et al. (1989) reached 19g X / l. Growth to higher cell densities required a fed-batch technique to eliminate both inhibition and limitation of substances and prevent the formation of inhibitory by-products of metabolism. In all cases it was necessary to dose other nutrients in addition to the C source and ammonia as N source and pH regulator. Using a glucose-mineral salt medium, Eppstein et al. (1989) by additional replenishment of salts 39g X / l. Fass et al. (1989) achieved 45g X / l by adding glucose solution containing magnesium sulfate to a glucose-mineral salt medium presented. Additional replenishment of salts resulted in Pan et al. (1987) to 95g X / l. Other fed-batch fermentations were carried out in glucose mineral salts containing yeast extract or autolyzed yeast powder prior to inoculation. Without additional feeding during cell growth, except for glucose and ammonia, 38 g X / l were obtained (Mori et al., 1979). Additional dosing of salts gave higher biomass concentrations of 47 g X / l (Bauer and White 1976), 55 g X / l (Shiloach and Bauer 1975) and 68 g X / l (Bauer and Ziv 1976). Additional dosing of salts, trace elements and vitamins led to 70g X / l (Fieschko and Ritsch 1986). Very high cell densities of 110 g X / L (Cutayar and Poillon 1989) and 125 g X / L (Mori et al., 1979) could be obtained by complex replenishment of salts, yeast extract and trace elements in addition to the usual dosage of glucose and ammonia. Further supplementation of a glucose mineral salt medium containing yeast extract prior to inoculation with bactotryptone or peptone and addition of these complex substrates during fermentation did not further increase the biomass concentration (Bailey et al 1987, Tsai et al 1987). Very high biomass concentrations of 80 to 105 g X / l (Eppstein et al., 1989) were achieved by combining the above-described fed-batch technique (metering of glucose and salts) by fumigation with oxygen-enriched air. Krüger (1989) achieved 112 g X / l by fumigation with pure oxygen.

The above-described methods all suffer from the disadvantage that they represent very complex refeeding systems. The addition of any additional nutrients other than glucose or magnesium sulfate-added glucose and ammonia requires special nutritional substrate dosages that involve either temporary additions or timed addition phases. This also relates to the use of the gaseous substrate oxygen.

A departure from such complex replenishment systems was then for the first time with the method for E. coli high cell density culturing of T.MATSUI et al. (Agric Biol Chem. 1989, 53, 8, 2115-20; Pat. No. 63,157,974), ie, in connection with this method solution, it was possible for the first time to indicate a (starting) nutrient medium with sufficiently complete mineral salt and trace metal ion properties so that now fermentations lead to high cell densities, with the sole addition of glucose and ammonium water feasible (up to biomass concentrations of 134g X / l). However, with this method, no longer fed-batch fermentation sections with a constant specific growth rate μ <M _mix can be realized (see the statements below), which does not seek any feedback control of this state parameter. For the process of MATSUI et al. is also disadvantageous that the carbon source is nachzufüttern in the form of powder, which requires the use of specially manufactured dosage technique. From the point of view of its overall effectiveness, this high-cell-density method still leaves something to be desired, since the mineral salt and trace metal ions are to be provided in extremely high initial concentrations, and during the fermentation the transition to fumigation with oxygen is unavoidable.

E. coli cells, which serve as hosts for vectors and thus used to form recombinant DNA products, must be adequately fermented to the recombinant product expression system to be used. This means that E.coli hosts are first fermented with connectable expression systems to high cell densities and then the connection of the recombinant DNA expression is made. For E.coli are also a number of homologous and heterologous expression systems that are constitutive or in which the expression at low specific growth rates is higher than at the maximum in each nutrient medium possible specific growth rate M _m "known. The specific growth rate is defined as follows:

There are some known solutions with which a growth of £. coli with μ <p _{mlx was} realized. Thus orreichten Zabriskie and Arcurj (1988) by the feeding of the carbon source to rermentationslösung at a constant rate, which was less than that which would have led to \ i _m "means that the culture with μ <\ i _{m, x} grew. Disadvantage of this procedure, however, is that μ constantly changes. If the dosage rate was kept constant over the entire fed-batch process, μ would constantly fall. Allen and LuIi (1985) developed a strategy to temporarily reset the specific growth rate μ with increasing cell density from time to time by adjusting new carbon source feed rates. This strategy implies the disadvantage that μ varies constantly within a tolerance range that depends on the frequency of off-line measurements of glucose and cell density, although growth with μ <Mm _{1x is} enforced. Changes in the specific growth rate result in discontinuities in the metabolism and can have a destabilizing effect, especially when it comes to the formation of inhibitory by-products of metabolism.

To achieve constant μ growth, Lee and Monier (1989) presented a controlled growth rate fermentation. The core of the procedure is the constant measurement of the carbon dioxide formed by the cells during growth, the respective current growth rate calculated by computer and the immediate realization of the adjustment of the dosing rate of the carbon source with the coupled feeding of C necessary to maintain the target value growth rate -Source. In this way, Lee and Mohler (1989) succeeded in cultivating E. coli with approximately constant μ <M _m "in the range of cell densities of 0.3 to 60 g X / l (EP 0315944 A2). The strategy of keeping μ constant over the measured variable CO], however, in principle has the disadvantage that CO _{2 is} not a growth-correlated signal in all cases. This is especially true when metabolism changes take place or anaplerotic biosynthetic pathways are used by the cells, which can occur especially at very high cell densities and carbon deficiency. Another disadvantage of the approach of Lee and Mohler is that after exhausting all possibilities to increase the oxygen input in the fermenter system used in each case, the growth breaks off quickly, since the dissolved oxygen concentration drops immediately. After the growth _phase with μ = const <M _mlx , a noticeable prolongation of the growth to further increase the cell density with μ <p _mlx with constantly decreasing μ is thus not possible. Furthermore, the dissolved carbon dioxide concentration in the adjusted pH range is very much dependent on the pH; Small pH changes have a negative effect on the measurement signal and can lead to a falsification of the μ calculation.

Object of the invention

The invention aims to ferment E. coli populations in stirred tank fermentors with more effective substrate utilization to very high cell densities.

Explanation of the essence of the invention

It is an object of the present invention to describe an inexpensive process by which E. coli strains can be fermented in stirred tank fermentors with effective substrate use to high cell densities. According to the invention, this object is achieved by a method for high-cell density fermentation of Escherichia coli in Rührkesseifermentor, which is characterized in that one fermented in a medium containing nitrogen source, organic carbon source and mineral salts and the fermentation is carried out in sections, by

first a batch section with maximum specific growth rate and

- thereafter provides a fed-batch section with submaximal oxygenation controlled and / or regulated specific growth rate, in which section glucose solution supplemented with magnesium sulfate is added.

According to a specific embodiment, in the fed-batch section, an approximately constant submaximal specific Provide growth rate. Further, the Fed-Batch section after reaching the by the Rührkesseifermentor

predetermined maximum oxygen input to be terminated with a subsection in which the specific

Growth rate drops. Since 26.11.1973 the German Collection of Microorganisms and Cell Cultures has filed a deposit for the Strain E. coli K12 (Reg. No. DSM498); the strain E. coli TG1, however, is previously published in Carter et al. (1985), Nucleic Acids

Res. 13: 4431-4443.

It is possible to carry out the fermentation in the presence of supplines and thereby compensate for auxotrophies. Is one Thlamin auxotrophy, so you can ferment the E. coli strain in the presence of thiamine. For example, you can Ferment E. coli TG1 in the presence of a thiamine concentration of 4.5mg / l. It is possible to supply the gaseous oxygen only with the aid of air during the entire fermentation. One can

However, the gaseous oxygen also first with the help of air and then with oxygen-enriched air or

Use oxygen. As an organic carbon source, it is preferable to use monosaccharides, disaccharides and / or polyols, glucose, Sucrose, lactose and / or glycerin. According to a specific embodiment of the proposed method

It is possible to use glucose solution supplemented with magnesium sulfate (for example a concentration of 19.2 g MgSO ₄ .7H ₂ O / 1) in the fed-batch section (for example a concentration <700 g / l) and optionally anti-foaming agent.

According to another specific embodiment, the nutrient medium used in the proposed method can already

contain all the nutrient substrates for the entire fermentation with the exceptions that

- aqueous ammonia solution (for example, a concentration of 25%) is metered in for pH adjustment,

- in the fed-batch section with magnesium sulfate (for example, a concentration of 19.2 g MgSO ₄ · 7H ₂ OZI) supplemented glucose solution (for example, a concentration of 700g l / l) is added and

- If necessary, anti-foaming agent, for example Ucolub N115 (Brenntag Mineraloel GmbH, FRG), is added,

The nutrient medium used in the proposed method may qualitatively include, for example, the following components

include:

Glucose, potassium dihydrogen phosphate, diammonium hydrogen phosphate, magnesium sulfate, iron citrate, cobalt chloride, Manganese chloride, copper chloride, BorsBure, sodium molybdate, zinc acetate, Titriplex III (ethylenediaminetetraacetic acid)

disodium salt; Merck, BRO) and / or citric acid and, if necessary, antifoaming agents and optionally supplements for the compensation of

Auxotrophy. In particular, the nutrient medium used can comprise, for example, the following components: glucose (s 50 g / l), KH ₂ PO ₄

(sM3.3g / l), <NH ₄ ) ₂ ) HPO ₄ (S4g / I), MgSO ₄ .7H ₂ O (s 1.2g / l), iron citrate (seOmg / l), CoCI ₂ .6H ₂ O ("52.5 mg / l), MnCl ₂ · 4H ₂ Q (s: i5 mg / l), CuCl ₂ · 2H ₂ O (S1.5 mg / l), H ₃ BOj (< ₃ mg / l), Ne ₂ Mo0 ₄ · 2 H ₂ O (S2,5mg / I), Zn (CH ₃ COO) ₂ · 2H ₂ O (: £ 8mg / l), Titriplex III (£ 8.4 mg / l) and / or citric acid (S 1.7 g / l) and, if necessary, anti-foaming agent ubcolub N115 (Ł1.1 ml / l).

To carry out the proposed method, the components of the nutrient medium in the following order

bring in the Fermeiitor:

first potassium dihydrogen phosphate, diammonium hydrogen phosphate and / or citric acid;

thereafter a solution of cobalt chloride, manganese chloride, copper chloride, boric acid, sodium molybdate, zinc acetate and / or Titriplex III, optionally prepared from stock solution,

- then iron citrate,

- Then if necessary, an antifoam agent, after which sterilized.

For the solution prepared from stock solutions one can first submit Titriplex III. < After sterilization of the solution presented in the fermentor can be a sterilized solution of glucose and / or Add magnesium sulfate to the fermentor. After inoculation of the nutrient medium, a lag phase may occur before the batch section. In the batch section can be, for example, at a pH s 7.5, in particular in the range of 6.6 to 6.9 and preferably at

about 6.8, ferment. The adjustment of the pH can be carried out with aqueous ammonia solution (a concentration of, for example, S 25%).

According to a specific embodiment of the proposed method, the batch section can be characterized by gradual Increase the stirrer speed a pO ₂ 2 1%, preferably in the range of 5 to 20% and in particular about 10% adjust. At the end of the batch section, the submaximal specific growth rate of the fed-batch section can be determined

go over that one

either the stirrer speed is lowered, for example in the range from 5 to 60 min,

- Or the stirrer speed keeps constant, for example in the range of 0.5 to 10 h.

In the Fed-Batoh section can be with the help of glucose dosage a pO ₂ a 1%, preferably in the range of 5 to 20% and

in particular of about 10%.

Also, one can use the fed-batch section to increase the desired submaximal specific growth rate Adjust oxygen input. This can be the stirrer speed, the gassing rate, the pressure and / or the oxygen content

increase the air or gas with oxygen.

By monitoring the submaximal specific growth rate at the fed-batch section, you can use one

Set adequate oxygenation. The submaximal specific growth rate can preferably be determined by means of the

Monitor the oxygen content of the fermentor exhaust air. According to a specific embodiment of the proposed method, the culture in the fed-batch section,

after the maximum technically achievable oxygen entry has been achieved, using the pO _{2 control} by

Continue to ferment glucose dosage with decreasing submaximal specific growth rate. For the sake of completeness it should also be noted that the concentration of dry biomass can be varied by

the ratio of the duration of the batch section to the duration of the fed batch section and / or

- In the fed-batch section, the height of the approximately constant submaximal specific growth rate varies. Thus, at least about 95 g X / l, for example, when culturing E. coli TG1 at μ = 0.107 1 / h = const, in the fed-batch section can be achieved. '

Surprisingly, once the oxygen input rate can not be increased for the particular fermentor system, the present process allows further growth; but with decreasing μ without lack of oxygen for the cells in the culture solution by means of the present pO _{2 control} . In the medium described above, a biomass dry matter concentration of at least 110 g X / l was obtained in this way with E. coli TG1, with the final volume not exceeding 76% of the fermentor ferretovolume

 The invention will be explained in more detail by a figure and an embodiment.

embodiment

The high cell density ferrination method will be described in more detail using the example of the cultivation of E. coli TG1 in the 72 l fermentor (B. Braun Melsungen AG, type 8015.1.01).

The broth to be inoculated from 37I contained the following components: glucose (2C-j / l), KH ₂ PO ₄ (13.3 g / l), (NH ₄ J ₂ HPO ₄ ( ₄ g / l), MgSO ₄ .7H ₂ O (1.2g / l), iron (III) citrate (60mg / l), CoCI ₂ 6H ₂ O (2.5 mg / l), MnCl ₂ .4H ₂ O (15mg / l), CuCl ₂ 2H ₂ O (1.5 mg / l), H ₃ BO ₃ ( ₃ mg / l), Na ₂ MoO ₄ .2H ₂ O (2.5 mg / l), Zn (CH ₃ COO) ₂ .2H ₂ O (8 mg / l), thiamine (4.5 mg / l), Titriplex III (8.4 mg / l), citric acid (1.7 g / l) and Ucolub N115 (0.1 ml / l) The preparation of this nutrient solution was carried out as follows: Initially, 148 g (NH ₄ I ₂ HPO ₄ , 492.1 g KH ₂ PO ₄ and 62.9 g citric acid as dry substance were dissolved in about 301 deionized water in the fermenter, followed by the addition of 257.4 ml trace mixture solution, the addition of a Iron (III) citrate solution (2.22 g iron (III) citrate in 300 ml) and 3.7 ml Ucolub N115 The trace mixture solution was prepared from stock solutions in the following

Manner: 62,2ml Tltrlplex III (6g / l), 34,3ml CoCI ₂ .2H ₂ O (2.7g / l), 34,7ml CuCl ₂ .2H ₂ O (1.6g / l), 34, 7 ml of MnCl ₂ · 4H ₂ O (16 g / l), 27.8 ml of H ₃ BO ₃ ( ₄ g / l), 30.8 ml of Ne ₂ MoO ₄ · 2H ₂ O (3 g / l) and 32.9 ml of Zn (CH ₁ COO) ₂ x 2H ₂ O (9 g / l). The solution In the fermentor was sterilized in the usual way. Then separately sterilized glucose solution (1.0175 kg glucose monohydrate in 2.5 l of H ₂ O), sterilized magnesium sulfate solution (44.5 g MgSO ₄ .7H ₂ O in 11H ₂ O), sterile-filtered thiamine solution (166.5 mg in 10OmI H ₂ O) was added, the pH adjusted to 6.8 with aqueous ammonia solution (25%), and finally filled up with sterile H ₂ O until reaching 36.81. Subsequently, the inoculation with 20OmI of a thawed Glycerln-Kulturvererve (20% v / v) of E. coli TG1.

Cultivation of E. coli TG1 was carried out at a temperature of 28 ^° C., a pressure of 1.5 bar, a pH of 6.8 and a gassing rate of 85 l / min. The pH was kept constant throughout the fermentation by controlled dosing of 25% NH ₃ . Fig. 1 shows the time courses of the stirrer speed, the concentration of the dry biomass X, the glucose concentration and the dissolved oxygen concentration. In the high-cell-density fermentation batch section the culture grew after the inoculation at time t = O, after a short lag phase of approximately 2h with maximum specific growth rate (p _m "x = 0.456 1 / h). After reaching the value of 10%, the pO _{2 was} kept constant by controlled increase of the stirrer speed in the further course. After consumption of initially submitted glucose, the batch section was completed. The PO ₂ increased drastically. Thereafter, no further regulation of the pO _{2 took place} via the stirrer speed. To reduce the specific growth rate of p _mu = 0.456 to μ = 0.11 1 / h, the stirrer speed was lowered from 420 rpm to 250 rpm for a period of 30 minutes. Thereafter, the pO _{2 control was started} via the dosing of glucose solution (700 g / l) supplemented with magnesium sulfate (19.2 g MgSO ₄ .7H ₂ O / I). The constant maintenance of μ In phase 1 of the fed-batch section was carried out by increasing the oxygen input. For this purpose, the speed was set via a PI controller with the controlled variable μ. The specific growth rate was determined indirectly from the oxygen balance of the system according to the following formula:

μ (ΰ

K + / Q ₀₂ (r) dr to

where: Qoj the oxygen consumption rate and

K is the quotient of the dry biomass concentration at time to and the yield coefficient for oxygen.

At the end of the first phase of the fed-batch section, a bio-dry matter concentration of 95 g / l was achieved. Due to the no longer increasing oxygen input into the culture solution after reaching the maximum technically possible stirrer speed, the culture in phase 2 of the fed-batch section then grew with constantly decreasing μ. At the end of this phase and thus to fermentation completion, a dry biomass of 110 g X / l could be obtained.

literature

Allen, B.R., and LuIi, G.W. (1986) A gradient-feed process for obtaining high cell densities for recombinant E.coll. Quoted in Lee and Monier: EP 0315944 A2

Bailey, F.J., Blankenship, J., Condra, J.H., Malgetter, R.Z., Ellis, R.W. (1987) High-cell density fermentation studies of a recombinant E.coll that expresses atrial natrluretic factor. J. Industrial Mlcroblol. 2: 47-52

Bauer, S., Shiloach, I. (1974). Maximum exponential growth rate and yield of E. coli obtainable in a bench-scale fermentor. Blotechn. Bioeng. 16: 933-941

- Bauer, S., While, M.D. (1976) Pilot scale exponential growth of Escherichia coli to high cell concentration with temperature variation. Blotechn. Bioeng. 18: 839-846

- Bauer, S., Ziv, E. (1976) Dense growth of aerobic bacteria In a bench-scale fermentor. Biotech. Bioeng. 18: 81-94

- Cutayar, J.M., Poillon, 0. (1989) High cell density culture of E.coli ana fed-batch system with dissolved oxygen as substrate feed indicator. Biotechnol.Lett.il: 155-160

- Eppstein, L, Shevitz, J., Yang, X.M., and Weise, S. (1989) Increased biomass production in a benchtop fermentor. Bio / Technology 7: 1178-1181

Fass, R., Clem, T.R., Shiloach, J. (1989) Use of a novel air separation system in a fed-batch fermentative culture of Escherichia coli. Appl. Environ. Microbiol. 65: 1305-1307

- Fleschko, J., Ritsch, T. (1986) Production of human alpha consensus interferon in recombinant Escherichia coil. Chem. Eng. Comm. 45: 229-240

Gleiser, I.E., Bauer, S. (1981) Growth of E. coli to high cell concentration by oxygen level linked control of carbon source concentration. Biotech. Bioeng. 23: 1015-1021

Jung, G., Denefle, P., Becquart, J., Mayaux, J.F. (1988) High cell density fermentation studies of recombinant Escherichia coli strains expressing human interleukin-1β. Ann. Inst. Pasteur / Microbiol. 139: 129-146

- KrOger, B. (1989) Yield increase in fermentation by computer use. BioTec Meßtechnik, Oct. 65-67

- Lee, S., and Mohler, R.D. (1989) Controlled growth rate fermentation. EP 0315944 A2

- Mizutani, S., Mori, H., Shlmlzu, S., Sakaguchi, K., Kobayashi, T. (1986) Effect of amino acid supplementation on cell yield and gene product In Escherichia coli harboring plasmid. Biotech. Bioeng. 28: 204-209

- Mori, H., Yano, T., Kodayashl, T., Shimizu, S. (1979) High Density Cultivation of Biomass In fed-batch system with OO-stat. J. Chem. Eng. 12: 313-319

- Pan, J.G., Rhee, J.S., Lebeault, J.M. (1987) Physiological contraints in increasing biomass concentration of Escherichia coli B In fed-batch culture. Biotechnol. Lett. 9: 89-94

- Reiling, H.E., Laurila, H., Flechter, A. (1985) Mass culture of Escherichia coli: Medium development for low and high density cultivation of Escherichia coli B / r in minimal and complex media. J. Biotechnol. 2: 191-206

- Shiloach, J., Bauer, S. (1975) High-yield growth of E.coli at different temperature In a bench scale fermentor. Biotech. Bioeng. 17: 227-239

- Tsa), LB, Mann, M., Morris, F., Rotgers, C, Fenton, D. (1987) The effect of organic nitrogen and glucose on the oroduction of recombinant human insulin-like growth factor in high cell density Escherichia coli fermentation. J. Industrial Microbiol. 2: 181-187

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Claims (29)

1. A process for the high-cell density fermentation of Escherichia coli in a stirred tank fermenter, characterized in that fermented in a medium containing a nitrogen source, organic carbon source and mineral salts and the fermentation is carried out in sections, by - first a batch section with maximum specific growth rate as well - thereafter provides a fed-batch section with submaximal oxygenation controlled and / or regulated specific growth rate, in which section glucose solution supplemented with magnesium sulfate is added. 2. The method according to claim 1, characterized in that in the fed-batch section provides a temporal profile for the submaximal specific growth rate. 3. The method according to claim 2, characterized in that it provides an approximately constant submaximal specific growth rate in the fed-batch section as temporal profile. 4. The method according to any one of the preceding claims, characterized in that one ends the fed-batch section after reaching the predetermined by the Rührkesselferrfientor maximum oxygen input with a subsection in which the submaximal specific growth rate decreases. 5. The method according to any one of the preceding claims, characterized in that one fermented E. coli K12 or a derivative thereof and preferably E. coli TG1. 6. The method according to any one of the preceding claims, characterized in that fermented in the presence of supplins and compensated for auxotrophies. 7. The method according to claim 6, characterized in that fermented at thiamine auxotrophy of the E. coli K12 strain to be fermented in the presence of thiamine. 8. The method according to claim 7, characterized in that one fermented E. coli TG1 in the presence of a thiamine concentration <4.5 mg / l. 9. The method according to any one of the preceding claims, characterized in that - supplies the gaseous oxygen only with the help of air during the entire fermentation or - At the beginning of the fermentation, the gaseous oxygen with air and later with oxygen-enriched air or pure oxygen feeds. 10. The method according to any one of the preceding claims, characterized in that one uses as organic carbon monosaccharides, disaccharides and / or polyols. 11. The method according to claim 10, characterized in that one uses as the organic carbon source of glucose, sucrose, lactose and / or glycerol. 12. The method according to any one of the preceding claims, characterized in that in the fed-batch section for feeding a solution of the above nature, the glucose in a concentration <700g / l and MgSo4 · 7HzO in a concentration <19.2 g / l contains, and optionally used anti-foaming agents. 13. The method according to any one of the preceding claims, characterized in that one uses at the beginning of a nutrient medium, which already contains all nutrient substrates for the total fermentation with the exception that in addition to the setting of claim 12 for pH adjustment aqueous ammonia solution is added. 14. The method according to any one of the preceding claims, characterized in that one uses a Närmedium, the qualitatively following components comprises: glucose, potassium dihydrogen phosphate, Diammoniumhydrogenphosphat, magnesium sulfate, iron citrate, cobalt chloride, manganese chloride, copper chloride, boric acid, sodium molybdate, zinc acetate, Titriplex IiI and / or citric acid and, if appropriate, anti-foaming agents and optionally supplements for compensating for auxotrophies. 15. The method according to claim 14, characterized in that one uses a nutrient medium comprising the following components: glucose (<50g / l). KH ₂ PO ₄ (<13.3 g / l), (NH ₄ J ₂ HPO ₄ (< ₄ g / l), MgSO ₄ .7H ₂ O (<1.2 g / l), iron citrate (<60 mg / l), CoCl ₂ · 6H ₂ O (<2.5 mg / l), MnCl ₂ · 4H ₂ O (<15 mg / l), CuCl ₂ · 2H ₂ O (<1.5 mg / l), H ₃ BO ₃ (< ₃ mg / l), Na ₂ MoO ₄ .2H ₂ O (<2.5 mg / l), Zn (CH ₃ COO) ₂ .2H ₂ O (<8 mg / l), Titriplex III (<8.4 mg / l) and or citric acid (<1.7 g / l) and, if necessary, anti-foaming agent UcolubN115 (<0.1 ml / l). 16. The method according to any one of the preceding claims, characterized in that one introduces the components of the nutrient medium in the following order in the fermentor: - first potassium hydrogen phosphate, diammonium hydrogen phosphate and / or citric acid; - Thereafter, optionally prepared from stock solutions of cobalt chloride, manganese chloride, copper chloride, boric acid, sodium molybdate, zinc acetate and / or Titriplex III, - then iron citrate, - then, if necessary, anti-foaming agent
and then sterilized. 17. The method according to claim 16, characterized in that initially submitted for the solution prepared from stock solutions Titriplex III. 18. The method according to any one of claims 11 to 17, characterized in that after sterilization of the solution introduced in the fermenter introduces a sterilized solution of glucose and / or magnesium sulfate in the fermentor. 19. The method according to any one of the preceding claims, characterized in that in the batch section at a pH <7.5, in particular in the range of 6.6 to 6.9 and preferably at about 6.8, fermented. 20. The method according to claim 13 and 19, characterized in that one uses for adjusting the pH aqueous ammonia solution of a concentration <25%. 21. The method according to any one of the preceding claims, characterized in that one sets in the batch section by means of gradually increasing the stirrer speed a pO ₂ ^ 1%, preferably in the range of 5 to 20% and in particular about 10%. 22. The method according to any one of the preceding claims, characterized in that one passes after completion of Fed-ätcr, -Abschnitt to submaximal specific growth rate of the fed-batch section by - either for a period of 5 minutes to 60 minutes, the stirrer speed is lowered - or keeps the stirrer speed constant for a period of 0.5 hours to 10 hours. 23. The method according to one of the preceding claims, characterized in that one sets in the fed-batch section with the aid of the glucose dosage a pO ₂ ^ 1%, preferably in the range of 5 to 20% and in particular of about 10%. 24. The method according to any one of the preceding claims, characterized in that adjusting the desired submaximal specific growth rate in the fed-batch section using increased oxygen input. 25. The method according to claim 24, characterized in that one increases the stirrer speed, the gassing rate, the pressure and / or the oxygen content of the air or with oxygen beg a sta. 26. The method according to any one of the preceding claims, characterized in that the submaximal specific Wachtumsrate monitored in the fed-batch section and rmt adjusts an adequate oxygen input of their help. 27. The method according to claim 26, characterized in that one monitors the submaximal specific growth rate with the aid of the oxygen content of the fermentation air. 28. The method according to any one of the preceding claims, characterized in that in the fed-batch section, after the maximum technically achievable oxygen input has been reached, the culture using the pOj control by glucose dosage weiterfermentiert with decreasing submaximal specific growth rate , 29. The method according to any one of the preceding claims, characterized in that one varies the concentration of dry biomass by the ratio of the duration of the batch section to the duration of the fed batch section and / or - In the fed-batch section, the level of the submaximal specific growth rate to be kept constant varies. For this 1 page drawing