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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor

Definitions

• the highly concentrated feeding solution is added continuously, whereby different functions can be used that define the addition of the substrate solution over time, for example, the addition takes place at a constant rate, increasing exponentially, or linearly increasing or decreasing. Different functions are often combined within a process.
• the nutrient solution is added in the form of pulses or intervals, the signal for the next pulse being, for example, the consumption of the nutrient or falling below a certain concentration of the nutrient (e.g. Terasawa et al., 1990, EP 0 397 097 AI).
• the addition of the substrate solution can also be regulated via other parameters.
• the control parameters used here are, for example, dissolved oxygen (DO-stat), pH (pH-stat), or the concentrations of carbon dioxide and oxygen in the exhaust gas determined on-line (e.g. Kerns et al., Acta Biotechnol. 8, 285 -289), which results in a cyclical dosing of the nutrient solution.
• the concentration of the substrate is varied between a limiting and a non-limiting concentration. Chen et al. (1997, Biotechnol. Bioeng. 56, 23-31) have measured an increased plasmid stability when periodically adding highly concentrated medium to the fed-batch culture. In these processes, a cycle spans several minutes or hours, which, however, had a negative effect on product formation.
• plasmids which, in addition to the origin of replication, contain at least the DNA sequence (product gene) coding for the desired protein and a selection marker which is intended to ensure the stable maintenance of the plasmid over the course of the culture.
• the expression of the product gene is usually controlled via regulatory sequences, in particular via regulatable promoters.
• the expression of the product gene is activated, for example, by chemical inducers (substrates, substrate analogs), the change in the cultivation temperature or other culture conditions (pH value, salt concentration, level of the substrate concentration).
• the induction can also take place by changing the limiting substrate, for example by inducing the tac promoter with lactose and switching from glucose feeding to lactose feeding (Neubauer et al., 1992, Appl. Microbiol. Biotechnol. 36, 739-744).
• genes serve as selection markers, which mediate resistance of the host cell to an antibiotic.
• the corresponding antibiotic which inhibits the growth of plasmid-free cells which do not carry the resistance gene, is then usually added to the culture for the production of a recombinant protein.
• resistance genes / antibiotic pairs are ß-lactamase / ampicillin, chloramphenicol acetyl transferase / chloramphenicol, tetracycline resistance (tet) -operon / tetracycline, kanamycin resistance gene / kanamycin.
• the antibiotic is inactivated by the resistance gene, such as, for example, ampicillin and chloramphenicol (for example Kemp GW and Britz ML, 1987, Biotechnol. Techniques 1, 157-162).
• This inactivation means that plasmid-free cells can multiply unhindered in the culture.
• the pre-culture can release the resistance-imparting proteins into the growth medium, which accelerate the breakdown of the antibiotic.
• the proportion of plasmid-free cells in the overall culture can be increased.
• no antibiotics are used for cost reasons or because of the additional effort that would be required in the subsequent cleaning, in which residual traces of the antibiotic or its inactivated form must also be removed used. Even with such processes, a certain proportion of plasmid-free cells is usually created.
• plasmid-free cells often only have a small growth advantage in the growth phase, in many cases after activation of the product formation, the growth rate of the plasmid-containing, producing cells is reduced and the culture is overgrown by the plasmid-free cell population.
• the accumulation of plasmid-free cells has the disadvantage that the relative proportion of the product in the total cell mass is reduced and, depending on the digestion and purification methods chosen, these steps following the fermentation are made more difficult.
• the invention specified in claim 1 is based on the problem of suppressing the growth of plasmid-free cells after induction of the recombinant product synthesis in fed-batch fermentations, in particular in the industrial sector, without a negative effect on product formation.
• the method is particularly suitable in fed-batch processes in which a sugar, such as. B. glucose, lactose, arabinose or galactose, or other organic carbon sources such as e.g. Methanol, glycerol, acetate, molasses or starch can be added to the culture as a limiting nutrient.
• a sugar such as. B. glucose, lactose, arabinose or galactose, or other organic carbon sources such as e.g. Methanol, glycerol, acetate, molasses or starch
• the process is independent of the cultivation medium and can be used for cultivation on mineral salt medium as well as on complex media.
• This method is not limited to Escherichia coli as the host organism, but can be used for all microorganisms, e.g. Bacillus subtilis, Saccharomyces cerevisiae or Pichia pastoris can be used, which are cultivated using carbon-limited fed batches. It is also independent of the induction system. However, it is particularly advantageous when using the tac promoter.
• the method is particularly advantageous when the expression of the gene product is strongly induced and the cell growth of the producing cells is negatively influenced in relation to a non-induced culture.
• This procedure also has advantages in processes in which the production phase is particularly long, for example in the periplasmic expression of recombinant proteins or when the product formation phase is associated with a temperature shift.
• Escherichia coli K-12 RB791 F, ⁇ N (rmD-rrnE) l, ⁇ ⁇ lad q L 8 ; E. coli Stock Center, New
• This strain was transformed with the plasmid pKK177glucC (Kopetzki et al., 1989a), in which the gene of the ⁇ -glucosidase from Saccharomyces cerevisiae is under the control of the tac promoter.
• the plasmid contains the ß-lactamase gene as a selection marker.
• a second system was used, into which, in addition to the plasmid pKK177glucC, the plasmid pUBS520 (Brinkmann et al, 1989) was transformed, which contains the dnaY gene (minor tRNA argU, AGA / AGG).
• Glucose-ammonium mineral salt medium (Teich et al., 1998, J. Biotechnol. 64, 197-210) was used for all cultivations.
• the starting concentration for glucose was 5 gl "1.
• Ampicillin (100 mg l "1 ) and kanamycin (10 mg l " 1 ) were added to both the precultures and the fermentation medium.
• Polypropylene glycol 2000 (50%) was used as an anti-foaming agent.
• Shake cultures on fermentation mineral salt medium, which were grown at 37 ° C., were used as the fermentation inoculum. All fermentations were carried out in 6 1 Biostat ED Bioreactor with a starting volume of 4 L and at a temperature of 35 ° C. The cultures were started as a batch culture. During this phase, the aeration rate and agitation were regulated in a cascade mode to keep the DOT at least 20%.
• the DOT control was switched off and the aeration rate and stirring speed were set to 2 wm and 800 rpm, respectively.
• the pH was adjusted to 7.0 using a 25% ammonia solution.
• the feeding pump was started at a constant rate of 53.2 gh " 1 (2.6 g glucose 1 "1 h " 1 ) , The total amount of glucose added was the same in all cultivations, regardless of the feed mode.
• Cell growth was monitored by measuring the optical density at 500 nm (OD 5 00). Furthermore, the microscopic number of cells in a counting chamber (0.02 mm depth) and the dry cell mass (DCW) were determined (see Teich et al., 1998, J. Biotechnol. 64, 197-210). The number of colony-forming units (cfu) was determined by spreading diluted samples on nutrient agar plates, which were incubated for at least 3 days. The plasmid stability was then determined by stamping these plates on selective agar using the replica plating technique.
• DCW dry cell mass
• DCW, OD 5 00 and cell number is characterized by the following relationship: lg / 1 DCW corresponds to an OD 50 o of 4.5 + 0.1 and a cell number of 1.8 ⁇ 10 9 ml “1.
• the glucose concentration was determined using a commercial enzyme kit.
• the ⁇ -glucosidase concentration was determined after separation of total cell samples in the SDS gel (5% stacking gel, 7% separation gel). Expression was determined by scanning the product band and quantifying it in relation to a product standard applied to the gel in different concentrations.
• E. coli RB791 pKK177glucC and E. coli RB791 pKK177glucC pUBS520 were cultivated by means of glucose-limited fed batch in a stirred reactor. After the first batch phase, constant feeding was started and three hours after the start of feeding the expression of the ⁇ -glucosidase gene was induced by adding 1 mM IPTG. After induction, there is an increase in the ⁇ -glucosidase concentration, the specific concentration of the enzyme per cell going through a maximum approx. 5 h after induction, but decreasing again with longer cultivation (see FIG. 1c). The decrease in the specific concentration of the ⁇ -glucosidase is due to the overgrowth of the culture with plasmid-free cells.
• Table 1 Productivity and overgrowth by plasmid-free cells in glucose-limited fed-batch cultures of E. coli RB791 pKK177glucC with and without pUBS520
• Fig. 1 Fed-batch fermentations with E. coli RB791 pKK177glucC pUBS520 with induction by 1 mM IPTG. Comparison of continuous addition of the glucose substrate solution (a-c; open symbols: without induction; filled symbols: with induction) with cyclical addition (d-f) of the same solution (A: cycle of 1 min; V: cycle of 4 min). (a, d) cell mass (DCW), (b, e) glucose concentration, (c, f) product formation (mg ⁇ -glucosidase / g dry cell weight). The data presented represent a characteristic fermentation of 2 experiments for continuous addition and 1 experiment for cyclic addition. Starting time for the addition of the substrate solution (), induction with IPTG took place at 3 h after feed
• Fig. 2 Fed-batch fermentations with E. coli RB791 pKK177glucC with induction by 1 mM IPTG. Comparison of continuous addition of the glucose substrate solution (a-c; open symbol: without induction; filled symbol: with induction) with cyclical addition (d-f) of the same solution (A: cycle of 1 min; V: cycle of 4 min). (a, d) cell mass (DCW), (b, e) glucose concentration, (c, d) product formation (mg ⁇ -glucosidase / g cell dry weight).

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