WO2001020016A2 - Method for increasing the yield of recombinant proteins in microbial fermentation processes - Google Patents

Method for increasing the yield of recombinant proteins in microbial fermentation processes Download PDF

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WO2001020016A2
WO2001020016A2 PCT/EP2000/008984 EP0008984W WO0120016A2 WO 2001020016 A2 WO2001020016 A2 WO 2001020016A2 EP 0008984 W EP0008984 W EP 0008984W WO 0120016 A2 WO0120016 A2 WO 0120016A2
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carbon
culture
energy source
induction
cycle
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PCT/EP2000/008984
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German (de)
French (fr)
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WO2001020016A3 (en
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Peter Neubauer
Hong Ying Lin
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Pharmacia Ab
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Priority to AU77763/00A priority Critical patent/AU775301B2/en
Priority to NZ517547A priority patent/NZ517547A/en
Priority to CA002383831A priority patent/CA2383831A1/en
Priority to IL14857500A priority patent/IL148575A0/en
Priority to KR1020027003256A priority patent/KR20020048934A/en
Priority to EP00967674A priority patent/EP1212450A2/en
Priority to JP2001523787A priority patent/JP2003530823A/en
Publication of WO2001020016A2 publication Critical patent/WO2001020016A2/en
Publication of WO2001020016A3 publication Critical patent/WO2001020016A3/en
Priority to HK03104274A priority patent/HK1052029A1/en

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    • C—CHEMISTRY; METALLURGY
    • 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

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  • 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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Abstract

The use of prior art methods for producing recombinant proteins in fed-batch fermentations often results, after the induction of the recombinant product synthesis, in an overgrowth of the culture due to plasmid-free cells and leads to a reduction of the specific product yield. The yield of recombinant proteins is thus increased by lowering or increasing, in a constantly brief manner, the concentration of the carbon/energy source in the culture. The oscillations are generated by altering the dosage rate of the feed solutions containing the carbon/energy source. This method is suited for all microorganisms which are cultivated using carbon-limited fed-batch.

Description

Verfahren zur Steigerung der Ausbeute von rekombinanten Proteinen in mikrobiellen Fermentationsprozessen Process for increasing the yield of recombinant proteins in microbial fermentation processes
Die Produktion rekombinanter Proteine in Bakterien erfolgt im industriellen Maßstab in Fermentoren. Eine Ertragssteigerung im Verhältnis zu Experimenten im Schüttelkolben- Labormaßstab wird über die Erhöhung der Zellmasse pro Volumen erreicht. Eine hohe Zelldichte kann durch die Fed-batch-Technik erzielt werden. Diese beruht auf der wachstumslimitierenden Zugabe einer Nährstoffquelle, wobei meistens die Kohlenstoff- und Energiequelle limitiert wird (z.B. Riesenberg D. and Guthke, R., 1999, Appl. Microbiol. Biotechnol. 51, 422-430). Bei E. co/7-Prozessen ist dies beispielsweise Glucose oder Glycerol. Alternativ werden in Abhängigkeit vom genutzten Mikroorganismus und vom Prozeß aber auch andere Substrate angewandt, wie beispielsweise Melasse, Stärke, Pepton, Laktose, Methanol und Acetat. Die Zugabe der hochkonzentrierten Feedinglösung erfolgt einerseits kontinuierlich, wobei unterschiedliche Funktionen genutzt werden können, die die Zugabe der Substratlösung über die Zeit definieren, beispielsweise erfolgt die Zugabe mit konstanter Rate, exponentiell ansteigend, beziehungsweise linear an- oder absteigend. Innerhalb eines Prozesses werden oft verschiedene Funktionen miteinander kombiniert. Alternativ erfolgt die Zugabe der Nährlösung in Form von Pulsen oder Intervallen, wobei beispielsweise das Signal für den nächsten Puls der Verbrauch des Nährstoffes oder das Unterschreiten einer bestimmten Konzentration des Nährstoffes ist (z.B. Terasawa et al., 1990, EP 0 397 097 AI). Die Zugabe der Substratlösung kann aber auch über andere Parameter geregelt werden. Hier verwendet man als Steuerparameter beispielsweise den Gelöstsauerstoff (DO-stat), den pH-Wert (pH-stat), oder die on-line ermittelten Konzentrationen von Kohlendioxid und Sauerstoff im Abgas (z.B. Kerns et al., Acta Biotechnol. 8, 285-289), wobei es zu einer zyklischen Dosierung der Nährlösung kommt. Dabei wird die Konzentration des Substrates zwischen einer limitierenden und einer nichtlimitierenden Konzentration variiert. Chen et al. (1997, Biotechnol. Bioeng. 56, 23-31) haben eine erhöhte Plasmidstabilität gemessen, wenn der Fed-batch-Kultur hochkonzentriertes Medium periodisch zugesetzt wird. Bei diesen Verfahren erstreckt sich ein Zyklus über mehrere Minuten bzw. Stunden, was sich jedoch negativ auf die Produktbildung auswirkte.The production of recombinant proteins in bacteria takes place on an industrial scale in fermenters. An increase in yield in relation to experiments on the shake flask laboratory scale is achieved by increasing the cell mass per volume. A high cell density can be achieved using the fed-batch technique. This is based on the growth-limiting addition of a nutrient source, whereby the carbon and energy source is usually limited (e.g. Riesenberg D. and Guthke, R., 1999, Appl. Microbiol. Biotechnol. 51, 422-430). In E. co / 7 processes, this is, for example, glucose or glycerol. Alternatively, depending on the microorganism used and the process, other substrates are also used, such as molasses, starch, peptone, lactose, methanol and acetate. On the one hand, 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. Alternatively, 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.
BESTATIGUNGSKOPIE Gängige Vektoren für die Genexpression sind Plasmide, die neben dem Replikationsorigin mindestens die für das gewünschte Protein kodierende DNA- Sequenz (Produktgen) sowie einen Selektionsmarker enthalten, der die stabile Erhaltung des Plasmids über den Kulturverlauf sichern soll. Die Expression des Produktgens wird üblicherweise über regulatorische Sequenzen, insbesondere über regulierbare Promotoren, gesteuert. Die Anschaltung der Expression des Produktgens erfolgt beispielsweise durch chemische Induktoren (Substrate, Substratanaloga), die Änderung der Kultivierungstemperatur oder anderer Kulturbedingungen (pH- Wert, Salzkonzentration, Höhe der Substratkonzentration). Insbesondere kann die Induktion auch durch einen Wechsel des limitierenden Substrates erfolgen, beispielsweise durch die Induktion des tac-Promoters mit Laktose und Umschaltung von Glucosefütterung auf Laktosefütterung (Neubauer et al., 1992, Appl. Microbiol. Biotechnol. 36, 739-744).BESTATIGUNGSKOPIE Common vectors for gene expression are 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). In particular, 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).
Für den stabilen Erhalt der Plasmide in den Wirtszellen dienen als Selektionsmarker Gene, die eine Resistenz der Wirtszelle gegenüber einem Antibiotikum vermitteln. In die Kultur zur Produktion eines rekombinanten Proteins wird dann üblicherweise das entsprechende Antibiotikum zugegeben, das das Wachstum von plasmidfreien Zellen, die das Resistenzgen nicht tragen, inhibiert bzw. diese abtötet. Häufig genutzte Resistenzgen/Antibiotika-Paare sind ß-Lactamase/Ampicillin, Chloramphenicol- acetyltransferase/Chloramphenicol, Tetracyclin-Resistenz (Tet)-Operon/Tetracyclin, Kanamycinresistenzgen/Kanamycin.For the stable maintenance of the plasmids in the host cells, 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. Frequently used resistance genes / antibiotic pairs are ß-lactamase / ampicillin, chloramphenicol acetyl transferase / chloramphenicol, tetracycline resistance (tet) -operon / tetracycline, kanamycin resistance gene / kanamycin.
Einige dieser Resistenzsysteme haben den Nachteil, daß das Antibiotikum durch das Resistenzgen inaktiviert wird, wie z.B. Ampicillin und Chloramphenicol (z.B. Kemp G.W. und Britz M.L., 1987, Biotechnol. Techniques 1, 157-162). Diese Inaktivierung hat zur Folge, daß sich plasmidfreie Zellen in der Kultur ungehindert vermehren können. Außerdem kann es schon bei der Vorkultur zur Freisetzung der die Resistenz vermittelnden Proteine in das Wachstumsmedium kommen, die den Abbau des Antibiotikums beschleunigen. In diesen Fällen kann der Anteil plasmidfreier Zellen an der Gesamtkultur noch erhöht sein. Weiter werden in einer großen Anzahl industrieller Prozesse aus Kostengründen oder wegen des zusätzlichen Aufwandes, der bei der nachfolgenden Reinigung anfallen würde, in der auch Restspuren des Antibiotikums beziehungsweise seiner inaktivierten Form entfernt werden müssen, keine Antibiotika eingesetzt. Auch bei solchen Prozessen entsteht meist ein bestimmter Anteil plasmidfreier Zellen.Some of these resistance systems have the disadvantage that 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. In addition, the pre-culture can release the resistance-imparting proteins into the growth medium, which accelerate the breakdown of the antibiotic. In these cases, the proportion of plasmid-free cells in the overall culture can be increased. Furthermore, in a large number of industrial processes, 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.
Obwohl plasmidfreie Zellen in der Wachstumsphase häufig nur einen geringen Wachstumsvorteil haben, kommt es nach Anschaltung der Produktbildung in vielen Fällen zu einer Verminderung der Wachstumsrate der plasmidhaltigen, produzierenden Zellen und damit zu einem Überwachsen der Kultur durch die plasmidfreie Zellpopulation. Die Anreicherung plasmidfreier Zellen hat den Nachteil, daß sich der relative Anteil des Produktes an der Gesamtzellmasse verringert und in Abhängigkeit von den gewählten Aufschluß- und Reinigungsmethoden diese der Fermentation nachfolgenden Schritte erschwert werden.Although 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.
Bei der Konstruktion des Vektors gibt es zwar die Möglichkeit, diese negativen Effekte einzuschränken, beispielsweise durch die Wahl des Resistenzgens, der Nutzung von alternativen, antibiotika-unabhängigen Stabilisierungssystemen (Molin und Gerdes, WO84/01172) oder durch die Nutzung modifizierter Antibiotika, die verlangsamt abgebaut werden, trotzdem werden die problematischen Resistenzen weithin eingesetzt. Außerdem ist keines der alternativen Systeme unendlich stabil und wird nur für einen gewissen Zeitraum stabil erhalten.When designing the vector, there is the possibility of limiting these negative effects, for example by choosing the resistance gene, using alternative, antibiotic-independent stabilization systems (Molin and Gerdes, WO84 / 01172) or by using modified antibiotics that slow down be dismantled, but the problematic resistances are widely used. In addition, none of the alternative systems is infinitely stable and is only kept stable for a certain period of time.
Der im Patentanspruch 1 angegebenen Erfindung liegt das Problem zugrunde, das Hochwachsen von plasmidfreien Zellen nach Induktion der rekombinanten Produktsynthese in Fed-batch-Fermentationen, insbesondere im industriellen Bereich, ohne negative Auswirkung auf die Produktbildung zu unterdrücken.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.
Dieses Problem wird durch die im Patentanspruch 1 angegebenen Merkmale gelöst, indem die Konzentration der KohlenstoffVEnergiequelle kurzzeitig zyklisch oszillierend abgesenkt bzw. erhöht wird. Dies wird durch die Veränderung der Zugaberate der die Kohlenstoff-/Energiequelle enthaltenden Fütterlösung erreicht, z. B. durch entsprechende Programmierung der die Fütterlösung zudosierenden Pumpe. Auf diese Weise entstehen aufeinanderfolgende Phasen, in denen die Zellen entweder Substrat in limitierter Form zur Verfügung haben, bzw. Substrathunger erfahren.This problem is solved by the features specified in claim 1 by briefly reducing or increasing the concentration of the carbon energy source in a cyclically oscillating manner. This is achieved by changing the rate of addition of the feed solution containing the carbon / energy source, e.g. B. by appropriate programming of the pump metering the feed solution. In this way, successive phases arise in which the cells either have limited substrate available or experience substrate hunger.
Entgegen bisheriger Auffassung, daß sich Oszillationen negativ auf die Produktbildung in rekombinanten Prozessen auswirken, können überraschenderweise gezielteContrary to the previous view that oscillations have a negative effect on the product formation in recombinant processes, surprisingly, targeted ones
Oszillationen, deren Zykluslänge maximal vier eine einzelne zyklische Phase maximal zwei Minuten beträgt, die Produktausbeute positiv beeinflussen. Besonders günstig sind Zykluszeiten einer Länge von ca. einer Minute (30 sec Feeding, 30 sec Pause).Oscillations whose cycle length is a maximum of four a single cyclic phase maximum is two minutes, positively affect the product yield. Cycle times of approximately one minute (30 sec feeding, 30 sec pause) are particularly favorable.
Bei dieser Verfahrensweise, die prinzipiell in allen rekombinanten wachstumslimitierten Prozessen angewandt werden kann, in denen die Bildung des rekombinanten Produktes unter Kohlenstofflimitation induziert wird, ist von Vorteil, daß kein Zusatz weiterer Substanzen zum Fermentationsmedium erforderlich ist, sie unabhängig vom genutzten Expressionssystem ist und sich nicht negativ auf die Produktbildung auswirkt. Besonders geeignet ist das Verfahren in Fed-batch Prozessen, bei denen ein Zucker, wie z. B. Glucose, Laktose, Arabinose oder Galaktose, oder andere organische Kohlenstoffquellen, wie z.B. Methanol, Glycerol, Acetat, Melasse oder Stärke als limitierender Nährstoff der Kultur zugesetzt werden. Dabei ist das Verfahren unabhängig vom Kultivierungsmedium und kann sowohl bei der Kultivierung auf Mineralsalzmedium wie auf Komplexmedien angewandt werden.In this procedure, which can in principle be used in all recombinant growth-limited processes in which the formation of the recombinant product is induced under carbon limitation, it is advantageous that no addition of further substances to the fermentation medium is necessary, it is independent of the expression system used and is not has a negative impact 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. The process is independent of the cultivation medium and can be used for cultivation on mineral salt medium as well as on complex media.
Diese Methode ist nicht auf Escherichia coli als Wirtsorganismus beschränkt, sondern kann bei allen Mikroorganismen, wie z.B. Bacillus subtilis, Saccharomyces cerevisiae oder Pichia pastoris, eingesetzt werden, die mittels kohlenstofflimitierten Fed-batch kultiviert werden. Sie ist auch unabhängig vom Induktionssystem. Jedoch ist sie bei der Nutzung des tac-Promoters besonders vorteilhaft.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.
Das Verfahren ist insbesondere dann vorteilhaft, wenn die Expression des Genproduktes stark induziert wird und das Zellwachstum der produzierenden Zellen im Verhältnis zu einer nichtinduzierten Kultur negativ beeinflußt wird. Weiterhin hat diese Verfahrensweise in Prozessen Vorteile, in denen die Produktionsphase besonders lang ist, beispielsweise bei der periplasmatischen Expression rekombinanter Proteine oder wenn die Produktbildungsphase mit einem Temperaturshift verbunden ist.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.
Ausführungsbeispielembodiment
Stamm und PlasmideStrain and plasmids
Escherichia coli K-12 RB791 (F, ιN(rmD-rrnE)l, λ\ ladqL8; E. coli Stock Center, NewEscherichia coli K-12 RB791 (F, ιN (rmD-rrnE) l, λ \ lad q L 8 ; E. coli Stock Center, New
Haven, USA) wurde als Wirt verwendet. Dieser Stamm wurde mit dem Plasmid pKK177glucC (Kopetzki et al., 1989a) transformiert, in dem das Gen der α-Glucosidase aus Saccharomyces cerevisiae unter Kontrolle des tac-Promoters steht. Das Plasmid enthält das ß-Lactamasegen als Selektionsmarker. Zusätzlich wurde ein zweites System verwendet, in das zusätzlich zu dem Plasmid pKK177glucC noch das Plasmid pUBS520 (Brinkmann et al, 1989) transformiert wurde, das das dnaY Gen (Minor- tRNA argU, AGA/AGG) enthält.Haven, USA) was used as the host. This strain was transformed with the plasmid pKK177glucC (Kopetzki et al., 1989a), in which the gene of the α-glucosidase from Saccharomyces cerevisiae is under the control of the tac promoter. The plasmid contains the ß-lactamase gene as a selection marker. In addition, 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).
Kultivierungsmedium und FermentationsbedingungenCultivation medium and fermentation conditions
Für alle Kultvierungen wurde Glucose-Ammonium-Mineralsalzmedium (Teich et al., 1998, J. Biotechnol. 64, 197-210) verwendet. Die Startkonzentration für Glucose lag bei 5 g l"1. Die Feed-Lösung enthielt 200 g Glucose kg"1 und alle Komponenten des Kultivierungsmediums in den entsprechenden Konzentrationen (Ausnahme (NH4)2SO4 2.0 g l"1) und 10 ml l"1 der Spurenelementlösung (Holme et al., 1970), aber kein MgSO . Dieses wurde im Verlauf der Kultivierung zu 10 ml einer 1 M MgSO Lösung per OD5oo=9 zugesetzt. Ampicillin (100 mg l"1) und Kanamycin (10 mg l"1) wurden sowohl den Vorkulturen als auch dem Fermentationsmedium zugesetzt. Polypropylenglycol 2000 (50 %) wurde als Antischaummittel genutzt. Als Fermentationsinokulum wurden Schüttelkulturen auf Fermentations- Mineralsalzmedium genutzt, die bei 37°C angezogen wurden. Alle Fermentationen wurden in 6 1 Biostat ED Bioreactor mit einem Startvolumen von 4 L und bei einer Temperatur von 35°C durchgeführt. Die Kulturen wurden als Batch-Kultur gestartet. In dieser Phase wurden die Belüftungsrate und die Rührung in einem Kaskadenmodus reguliert, um den DOT auf mindestens 20% zu halten. Am Ende der Batch-Phase wurde die DOT-Kontrolle abgeschaltet und Belüftungsrate und Rührgeschwindigkeit auf 2 wm bzw. 800 rpm gesetzt. Der pH- Wert wurde mittels 25 %iger Ammoniaklösung auf 7.0 geregelt. Am Ende der Batch-Phase, bei einer Zelldichte von ca. 2 g DCW l"1 (OD5oo=9) wurde die Feedingpumpe mit einer konstanten Rate von 53.2 g h"1 (2.6 g Glucose l"1 h"1) gestartet. Die Gesamtmenge zugesetzter Glucose war die gleiche in allen Kultivierungen, unabhängig vom Feed-Modus. Drei verschiedene Feedingstrategien wurden getestet: (A) kontinuierliches Feeding (Kontrollkultivierung), (B) unterbrochenes Feeding mit einem Zyklus von 1 Minute (30 Sekunden an, 30 Sekunden aus), (C) unterbrochenes Feeding mit einem Zyklus von 4 Minuten (2 Minuten an, 2 Minuten aus). Die Expression des α-Glucosidasegens wurde durch Zusatz von 1 mM IPTG 3 h nach Feedingstart induziert und die Produktbildung über einen Zeitraum von ca. 20 h nach Induktion verfolgt. Analytische MethodenGlucose-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. The feed solution contained 200 g glucose kg " 1 and all components of the cultivation medium in the corresponding concentrations (exception (NH 4 ) 2 SO 4 2.0 gl "1 ) and 10 ml l " 1 of the trace element solution (Holme et al., 1970), but no MgSO. In the course of the cultivation, this was added to 10 ml of a 1 M MgSO 4 solution using OD 5 oo = 9. 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%. At the end of the batch phase, 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. At the end of the batch phase, at a cell density of approx. 2 g DCW 1 "1 (OD 5 oo = 9), 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. Three different feeding strategies were tested: (A) continuous feeding (control cultivation), (B) interrupted feeding with a cycle of 1 minute (30 seconds on, 30 seconds off), (C) interrupted feeding with a cycle of 4 minutes (2 minutes on, 2 minutes off). The expression of the α-glucosidase gene was induced by adding 1 mM IPTG 3 h after the start of feeding and the product formation was monitored over a period of about 20 h after induction. Analytical methods
Das Zellwachstum wurde durch die Messung der optischen Dichte bei 500 nm (OD500) verfolgt. Weiterhin wurde die mikroskopische Zellzahl in einer Zählkammer (0.02 mm Tiefe), und die Zelltrockenmasse (DCW) bestimmt (siehe Teich et al., 1998, J. Biotechnol. 64, 197-210). Die Anzahl koloniebildender Einheiten (cfu) wurde durch Ausstreichen verdünnter Proben auf Nähragarplatten ermittelt, die mindestens 3 Tage bebrütet wurden. Die Plasmidstabilität wurde darauf durch Überstempeln dieser Platten auf Selektivagar mit der Replica plating- Technik bestimmt. Das Verhältnis zwischen DCW, OD500 und Zellzahl wird durch folgende Beziehung charakterisiert: lg/1 DCW entspricht einer OD50o von 4.5+0.1 und einer Zellzahl von 1.8xl09 ml"1. Die Glucosekonzentration wurde mit einem kommerziellen Enzymkit ermittelt.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. The relationship between 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.
Die Bestimmung der α-Glucosidasekonzentration erfolgte nach Auftrennung von Gesamtzellproben im SDS-Gel (5 % Sammelgel, 7 % Separationsgel). Die Expression wurde durch Scannen der Produktbande und Quantifizierung im Verhältnis zu einem jeweils auf dem Gel in verschiedenen Konzentrationen aufgetragenen Produktstandard.The α-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.
ErgebnisseResults
E. coli RB791 pKK177glucC und E. coli RB791 pKK177glucC pUBS520 wurden mittels glucoselimitiertem Fed-batch in einem Rührreaktor kultiviert. Nach der ersten Batchphase wurde ein konstantes Feeding gestartet und drei Stunden nach Feedingstart wurde die Expression des α-Glucosidasegens durch Zusatz von 1 mM IPTG induziert. Nach Induktion kommt es zu einem Anstieg der α-Glucosidasekonzentration, wobei die spezifische Konzentration des Enzyms pro Zelle ca. 5 h nach Induktion ein Maximum durchläuft, bei längerer Kultivierung aber wieder abnimmt (s. Fig. lc). Die Abnahme der spezifischen Konzentration der α-Glucosidase ist auf das Überwachsen der Kultur mit plasmidfreien Zellen zurückzuführen. Diese haben nach Induktion einen enormen Wachstumsvorteil, da sich die Produktion der α-Glucosidase negativ auf das Wachstum auswirkt und auch eine Inhibition der Glucoseaufnahme in den produzierenden Zellen bewirkt. Dies führt zu einer Anreicherung von Glucose im Kulturmedium. In der Kultur vorhandene Zellen, die das Produktgen nicht enthalten, werden durch den Induktor IPTG nicht beeinflußt, vielmehr wachsen sie durch die hohe Verfügbarkeit an Glucose unlimitiert weiter. Wenn die Glucoselösung nicht kontinuierlich, sondern pulsweise in kurzen Zyklen, die im Größenbereich einer Minute liegen, zugesetzt wird (siehe Material und Methoden), wird die α-Glucosidase in ähnlicher Weise wie beim konstanten Feed nach Induktion angereichert. Allerdings kann in Abhängigkeit von der Pulslänge das Überwachsen der Kultur durch die plasmidfreie Zellpopulation verhindert werden (siehe Fig. Id). Dieser positive Effekt auf die Unterdrückung plasmidfreier Zellen war nicht nur im in Fig. 1 dargestellten stark exprimierenden System offensichtlich, sondern auch bei der schwachen Expression der α-Glucosidase im System E. coli RB791 pKK177glucC (Fig. 2, Tabelle 1). Außerdem hatte das pulsweise Feeding in beiden Fällen einen leicht positiven Einfluß auf die Syntheserate nach Induktion und im ersten Fall auch auf die Stabilität des Produktes, das zu mehr als 90 % in Form von Einschlußkörpern (inclusion bodies) vorlag. Ein wichtiger Faktor ist die Definition der Zykluszeit. In beiden gezeigten Beispielen führt die Verlängerung der Zykluszeit auf 4 min zu einer Abnahme der Produktmenge und damit des Ertrages (siehe Abb. 1, 2 und Tabelle 1). Obwohl auch in diesem fall das Überwachsen der Kultur durch plasmidfreie Zellen vermindert wurde, führt die ländere Zykluszeit zu einer geringeren Produktsynthese bzw, zu einem verstärkten Abbau. 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 α-glucosidase gene was induced by adding 1 mM IPTG. After induction, there is an increase in the α-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 α-glucosidase is due to the overgrowth of the culture with plasmid-free cells. These have an enormous growth advantage after induction, since the production of the α-glucosidase has a negative effect on the growth and also an inhibition of the glucose uptake in the producing cells. This leads to an accumulation of glucose in the culture medium. Cells present in the culture which do not contain the product gene are not influenced by the inducer IPTG, rather they continue to grow indefinitely due to the high availability of glucose. If the glucose solution is not added continuously, but in pulses in short cycles in the size range of one minute (see material and methods), the α-glucosidase is enriched in a similar way to the constant feed after induction. Depending on the pulse length, however, the overgrowth of the culture can be prevented by the plasmid-free cell population (see FIG. Id). This positive effect on the suppression of plasmid-free cells was not only evident in the strongly expressing system shown in FIG. 1, but also in the weak expression of the α-glucosidase in the E. coli RB791 pKK177glucC system (FIG. 2, Table 1). In addition, in both cases the pulsed feeding had a slightly positive influence on the synthesis rate after induction and in the first case also on the stability of the product, which was more than 90% in the form of inclusion bodies. An important factor is the definition of the cycle time. In both examples shown, the extension of the cycle time to 4 minutes leads to a decrease in the amount of product and thus in the yield (see Fig. 1, 2 and Table 1). Although the overgrowth of the culture by plasmid-free cells was also reduced in this case, the rural cycle time leads to a lower product synthesis or to an increased degradation.
Tabelle 1 : Produktivität und Überwachsen durch plasmidfreie Zellen in glucoselimitierten Fed-batch-Kulturen von E. coli RB791 pKK177glucC mit und ohne pUBS520Table 1: Productivity and overgrowth by plasmid-free cells in glucose-limited fed-batch cultures of E. coli RB791 pKK177glucC with and without pUBS520
Figure imgf000009_0001
Figure imgf000009_0001
Die Figuren zeigen:The figures show:
Fig. 1: Fed-batch Fermentationen mit E. coli RB791 pKK177glucC pUBS520 mit Indukion durch 1 mM IPTG. Vergleich von kontinuierlicher Zugabe der Glucose- Substratlösung (a-c; offene Symbole: ohne Induktion; gefüllte Symbole: mit Induktion) mit zyklischer Zugabe (d-f) derselben Lösung (A: Zyklus vom 1 min; V: Zyklus vom 4 min). (a,d) Zellmasse (DCW), (b,e) Glucosekonzentration, (c,f) Produktbildung (mg α- Glucosidase/g Zelltrockengewicht). Die dargestellten Daten repräsentieren eine charakteristische Fermentation von 2 durchgeführten Experimenten für die kontinuierliche Zugabe und je 1 Experiment für die zyklische Zugabe. Startzeitpunkt für die Zugabe der Substratlösung ( ), Induktion mit IPTG erfolgte bei 3 h nach Feed-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 α-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
Start ( ).Begin ( ).
Fig. 2: Fed-batch Fermentationen mit E. coli RB791 pKK177glucC mit Induktion durch 1 mM IPTG. Vergleich von kontinuierlicher Zugabe der Glucose-Substratlösung (a-c; offenes Symbol: ohne Induktion; gefülltes Symbol: mit Induktion) mit zyklischer Zugabe (d-f) derselben Lösung (A: Zyklus vom 1 min; V: Zyklus vom 4 min). (a,d) Zellmasse (DCW), (b,e) Glucosekonzentration, (c,d) Produktbildung (mg α- Glucosidase/g Zelltrockengewicht). Weitere Erklärungen siehe Fig. 1. Fig. 3: Darstellung des Pumpenschaltschemas in einer Fermentation mit einem Zyklus von 1 min. Gezeigt ist ein kleiner Ausschnitt der Fermentation, die Reaktion des Gelöstsauerstoffs (DOT, %, — υ — ), sowie die Pumpenschaltung (0 = aus, 1 = an,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 α-glucosidase / g cell dry weight). For further explanations see Fig. 1. Fig. 3: Representation of the pump switching scheme in a fermentation with a cycle of 1 min. Shown is a small section of the fermentation, the reaction of the dissolved oxygen (DOT,%, - υ -), as well as the pump switching (0 = off, 1 = on,
)• ) •

Claims

Patentansprüche claims
1. Verfahren zur Steigerung der Ausbeute von rekombinanten Proteinen in mikrobiellen Fermentationsprozessen, dadurch gekennzeichnet, daß die Konzentration der Kohlenstoff-/Energiequelle in der Kultur in kurzen Zyklen oszillierend abgesenkt beziehungsweise erhöht wird.1. A method for increasing the yield of recombinant proteins in microbial fermentation processes, characterized in that the concentration of the carbon / energy source in the culture is reduced or increased in an oscillating manner in short cycles.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß die Oszillationen durch die Veränderung der Zugaberate der die Kohlenstoff-/Energiequelle enthaltenden Fütterlösung erzeugt werden.2. The method according to claim 1, characterized in that the oscillations are generated by changing the addition rate of the feed solution containing the carbon / energy source.
3. Verfahren nach Anspruch 1 und 2, dadurch gekennzeichnet, daß die Länge eines Zyklus maximal 4 Minuten und eine einzelne zyklische Phase maximal zwei3. The method according to claim 1 and 2, characterized in that the length of a cycle at most 4 minutes and a single cyclic phase at most two
Minuten andauert.Minutes.
4. Verfahren nach Anspruch 1 bis 3, dadurch gekennzeichnet, daß die Länge eines Zyklus eine Minute beträgt und eine einzelne zyklische Phase maximal 75 % der Zykluszeit andauert. 4. The method according to claim 1 to 3, characterized in that the length of a cycle is one minute and a single cyclic phase lasts a maximum of 75% of the cycle time.
5. Verfahren nach Anspruch 1 bis 3, dadurch gekennzeichnet, daß der Kultur die Kohlenstoff-Energiequelle so zugesetzt wird, daß die Zuflußrate der Substratlösung nur in bestimmten zeitlichen Abschnitten des Prozesses zyklisch variiert wird.5. The method according to claim 1 to 3, characterized in that the culture, the carbon energy source is added so that the inflow rate of the substrate solution is varied cyclically only in certain time sections of the process.
6. Verfahren nach Anspruch 1 bis 5, dadurch gekennzeichnet, daß die Zuflußrate durch zyklisches An- und Abschalten des Zuflusses der Fütterlösung kontrolliert wird. 6. The method according to claim 1 to 5, characterized in that the inflow rate is controlled by cyclically switching on and off the inflow of the feed solution.
7. Verfahren nach Anspruch 1 bis 6, dadurch gekennzeichnet, daß als Kohlenstoff- Energie-Substrat Glucose, Glycerol, Laktose, Arabinose, Galaktose, Methanol, Acetat, Melasse oder Stärke verwendet wird.7. The method according to claim 1 to 6, characterized in that glucose, glycerol, lactose, arabinose, galactose, methanol, acetate, molasses or starch is used as the carbon energy substrate.
8. Verfahren nach Anspruch 1 bis 7, dadurch gekennzeichnet, daß zur Induktion der Bildung des rekombinanten Produktes in Abhängigkeit vom eingesetzten Promoter der Kultur IPTG oder Indolylacrylsäure (IAA), oder wenn diese nicht bereits als8. The method according to claim 1 to 7, characterized in that for induction of the formation of the recombinant product depending on the promoter of culture used IPTG or indolylacrylic acid (IAA), or if these are not already
Energiequelle verwendet werden Laktose, Arabinose, Galaktose oder Methanol zugesetzt werden.Lactose, arabinose, galactose or methanol can be added as an energy source.
9. Verfahren nach Anspruch 1 bis 9, dadurch gekennzeichnet, daß zum Induktionszeitpunkt der Bildung des rekombinanten Produktes ein Temperaturshift erfolgt. 9. The method according to claim 1 to 9, characterized in that a temperature shift takes place at the time of induction of the formation of the recombinant product.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1132464A2 (en) * 2000-03-10 2001-09-12 Andreas Dr. Schmid Methods to improve the performance of a microbial system
EP1132464A3 (en) * 2000-03-10 2004-04-21 Andreas Dr. Schmid Methods to improve the performance of a microbial system
US6905870B2 (en) 2000-11-17 2005-06-14 Microbes, Inc. Microbial-induced controllable cracking of normal and branched alkanes in oils
WO2003029439A1 (en) * 2001-10-01 2003-04-10 Novozymes A/S Fermentation with cyclic pulse-pause feeding
US7855059B2 (en) 2001-10-01 2010-12-21 Novozymes A/S Fermentation with cyclic pulse-pause feeding

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