KR20020048934A - Method for increasing the yield of recombinant proteins in microbial fermentation processes - Google Patents

Abstract

Using prior art methods of producing recombinant proteins in batch-feed fermentation, the culture is overgrown by plasmid-free cells after recombinant product synthesis is induced, thereby reducing the yield of specific products. The yield of recombinant protein is enhanced by decreasing or increasing the concentration of carbon / energy source in the culture in a continuous simple manner. Vibration occurs by varying the rate of administration of a feed solution containing a carbon / energy source. This method is suitable for all microorganisms cultured using carbon limited batch-feeding methods.

Description

METHOD FOR INCREASING THE YIELD OF RECOMBINANT PROTEINS IN MICROBIAL FERMENTATION PROCESSES

Industrial production of recombinant proteins in bacteria occurs in fermenters. Enhancement of yield compared to laboratory scale experiments performed with a flask shaker is achieved by increasing cell mass per volume. Batch-fed techniques can achieve high cell density. This is based on the growth-limiting addition of nutrients, where the carbon / energy source is generally limited [see, eg, Riesenberg D. and Guthke, R., App. Microbiol. Biotechnol. 51, 422-430 (1999) ". For the Escherichia coli ("E. coli") process, this is usually glucose or glycerol. Alternatively, however, other substrates are used, such as molasses, starch, peptone, lactose, methanol and acetate, depending on the microorganism and process used. Highly concentrated feed solutions can be added continuously, allowing the use of various functions to limit, linearly increase or decrease the addition of the substrate over a period of time. Various functions are often combined in a single process. Alternatively, the nutrient solution may be added in pulses or at intervals of time by reducing the amount of nutrients or consuming nutrients below a predetermined concentration that acts as a signal for the next wave [eg , EP 0 397 097 A1 (1999) by Terasawa et al. The addition of the substrate solution can also be controlled using other parameters. Depleted carbon dioxide and oxygen concentrations determined by dissolved oxygen (DO-stat), pH (pH-stat), or on-line (eg, Kerns et al., Acta Biotechnol. 8, 285 -289 "] can be used as control data to drive periodic administration of nutrient solutions. In this case, the concentration of the substrate varies between limited and non-limiting concentrations. Chen et al. (See Biotechnol. Bioeng. 56, 23-31 (1997)) measured enhanced plasmid stability when highly concentrated media were added periodically to batch-feed cultures. In this procedure, one cycle can last for minutes or hours, which adversely affects product formation.

Standard vectors for gene expression are, in addition to the origin of replication, plasmids which generally contain DNA sequences encoding the desired proteins (product genes) as well as selection markers which serve to ensure stable preservation of the plasmid during culture growth. Expression of the product gene is generally controlled via regulatory sequences, in particular regulatory promoters. Expression of the product gene is activated by, for example, chemical inducers (substrate, substrate analogues) and changes depending on the culture temperature or other culture conditions (pH value, salt concentration, degree of substrate concentration). In particular, induction can also occur by altering restriction substrates or by inducing a tac-promoter with lactose and switching from a glucose feed to a lactose feed [Neubauer et al., Appl. Microbiol. Biotechnol. 36, 739-744 (1992)] Genes that provide host cells resistant to antibiotics serve as selection markers that stably preserve the plasmid in the host cell. In the culture, the corresponding antibiotics are generally added to stop or inhibit the growth of plasmid-free cells that do not carry the resistance genes A commonly used resistance gene / antibiotic pair is β-lactamase / ampicillin, In chloramphenicol-acetyltransferase / chloramphenicol, tetracycline resistance (tet) -operon / tetracycline and kanamycin Sex gene / kanamycin.

Some of these resistance systems have the disadvantage that antibiotics are inactivated by resistance genes, as is the case with ampicillin and chloramphenicol (see, eg, Kemp GW and Britz ML, Biotehnol. Techniques 1 , 157-162 (1987) ". As a result of this inactivation, there is no barrier to replication of plasmid-free cells in culture. In addition, proteins that mediate resistance can be released into the medium in preculture, accelerating the destruction of antibiotics. In such cases, the proportion of plasmid-free cells in the total culture can be enhanced. Moreover, no antibiotics are used in many industrial processes, either because of the high cost or subsequent cleaning operations (remaining traces of antibiotics or their inactivated forms must be removed). It costs money. In addition, certain proportions of plasmid-free cells appear in this process.

Although plasmid-free cells often have only a small growth advantage in the growth phase, in many cases, after product formation has begun, the growth rate of the resulting cells containing the plasmids is reduced so that the culture is overwhelmed by the plasmid-free cell population. Is grown. Accumulation of plasmid-free cells has the disadvantage of reducing the relative proportion of product in the entire cell population and making this post fermentation step more difficult depending on the chosen digestion and washing method.

When constructing vectors, for example, through the selection of resistance genes, through the use of alternative antibiotic-independent stabilization systems (WO 84/01172 to Moline and Gerdes), or more Slowly breaking modified antibiotics can be used to limit this adverse effect; Problematic immunity is still used. Moreover, there are no infinitely stable alternative systems; Stability can only be maintained for a certain period of time.

1 shows E. coli induced with 1 mM IPTG. Batch-feed fermentation by E. coli RB791 pKK177glucC pUBS520. Continuous addition of the glucose substrate solution (a-c; hollow sign: no induction; filled sign: derived) is compared with the periodic addition (d-f) (▲: 1 minute cycle; ▽: 4 minute cycle) of the same solution. (a, d) cell mass (DCW), (b, e) glucose concentration, (c, f) product formation (mg α-glucosidase / g cell dry weight). The data shows the characteristic fermentation of one experiment for each of the two experiments and the periodic additions performed for the continuous addition. The start time is when the substrate solution is added (------), and induction by IPTG occurs 3 hours after the start of the feed (····).

2 shows E. coli induced with 1 mM IPTG. Batch-feed fermentation by E. coli RB791 pKK177glucC. Continuous addition of the glucose substrate solution (a-c; hollow sign: no induction; filled sign: derived) is compared with the periodic addition (d-f) (▲: 1 minute cycle; ▽: 4 minute cycle) of the same solution. (a, d) cell mass (DCW), (b, e) glucose concentration, (c, d) product formation (mg α-glucosidase / g cell dry weight). See FIG. 1 for further description.

3 shows a pump bowl configuration in a fermentation of 1 minute cycle. Small compartments of fermentation are shown, with pump switching (0 = off, 1 = on, ---) as well as the reaction of dissolved oxygen (DOT,%, --- υ ---) do.

The invention set forth in claim 1 suppresses the overgrowth of plasmid-free cells after the synthesis of the recombinant product is induced in batch-feed fermentation, especially in industrial use, without adversely affecting product formation. Based on the problem.

The features listed in claim 1 solve this problem by increasing / decreasing the concentration of the carbon / energy source in a periodic vibration pattern. This is achieved by varying the rate of addition of the feed solution containing the carbon / energy source, for example by correspondingly programming the pump for administering the feed solution. This leads to subsequent phases in which cells have limited amounts or no substrate available to them.

In contrast to the previously known view that vibrating pattern feeding adversely affects product formation in the recombination process, the desired vibrations with a maximum cycle duration of 4 minutes and individual phases of cycles lasting up to 2 minutes surprisingly affect product yield. Has a positive impact. Particularly advantageous is a cycle period of about 1 minute (30 second supply, 30 minute interruption).

The advantage of this method, which is mainly applicable to all recombinant growth-limited processes in which the formation of recombinant products is induced under carbon limitation, is that there is no need to add additional materials to the fermentation medium, regardless of the expression system used, It does not have a harmful effect. This procedure is particularly suitable for batch-feeding processes in which sugars such as glucose, lactose, arabinose or galactose, or other organic carbon sources such as methanol, glycerol, molasses or starch are added to the culture as limiting nutrients. This procedure is independent of the culture medium and can be used for culturing on complex medium as well as inorganic salt medium.

This method works as a host organism. Not all coli, but rather all microorganisms cultured using carbon-limited batch-feeding, such as Bacillus subtilis , Saccharomyces cerevisiae or Peachia Pichia pastoris can be used. It is also independent of the induction system. However, this is particularly useful when using a tak-promoter.

This procedure is particularly useful when the expression of product genes is strongly induced and the growth of the resulting cells is adversely affected compared to uninduced cultures. This procedure is also advantageous in processes where the productive phase is particularly long, for example when the recombinant protein is expressed in the periplasm or the product formation phase is associated with a change in temperature.

Working mode

Strains and Plasmids

this. E. coli K-12 RB791 [F ⁻ , IN (rmD-rmE) 1, λ ⁻ , lacI ^q L _g ; Lee, New Haven, USA. Obtained from E. coli Stock Center]. This strain was transformed with plasmid pKK177glucC (Kopetzki et al., 1989a), in which the α-glucosidase gene from Saccharomyces cerevisiae was under the control of the Tak-promoter. This plasmid contains the β-lactamase gene as its selection marker. In addition, a second system in which plasmid pUBS520 (Brinkmann et al., 1989) containing the dnaY gene (Minor-tRNA argU , AGA / AGG) was transformed in addition to plasmid pKK177glucC was used.

Culture medium and fermentation conditions

Glucose-ammonium-inorganic salt medium (Teich et al., "J. Biotechnol. 64, 197-210 (1998)") was used for all cultures. The initial concentration of glucose was 5 g / l. The feed solution is 200 g / kg of glucose, all components of the culture medium at the corresponding concentration (exception: 2.0 g / l for (NH ₄ ) ₂ SO ₄ ), and 10 ml / l of trace element solution [Holem] Et al., 1970, but no MgSO ₄ . To this was added 10 ml of a ₁ M MgSO ₄ solution with an OD ₅₀₀ of 9 during the culture. Ampicillin (100 mg / l) and kanamycin (10 mg / l) were added to both the preculture and the fermentation medium. Polypropylene glycol 2000 (50%) was used as antifoaming agent.

Shake cultures on fermentation inorganic salt medium, grown at 37 ° C., were used as fermentation inoculum. All fermentations were performed at 35 ° C. with an initial volume of 4 L in a 6 L Biostat ED Bioreactor. The culture was started as a batch culture. In this phase, the aeration rate and agitation were adjusted in cascade mode to maintain a DOT of at least 20%. At the end of the batch, the DOT control was deactivated and the aeration rate and stirring speed were set to 2vvm or 800rpm. The pH value was adjusted to 7.0 with 25% ammonia solution. At the end of the batch, the feed pump was started at a constant rate of 53.2 g / h (2.6 g / l / h glucose) at a cell density of about 2 g DCW / l (OD ₅₀₀ = 9). The total amount of glucose added was the same in all cultures regardless of the mode of feeding. Three different feeding strategies were tested: (A) continuous feeding (controlled culture), (B) intermittent feeding of 1 minute cycle (30 seconds feeding, 30 seconds interruption), and (C) intermittent feeding of 4 minutes cycle (2 minutes). Supply, 2 minutes interruption). Three hours after the start of feeding, 1 mM IPTG was added to induce the expression of the α-glucosidase gene, and product was formed for the entire period of about 20 hours after induction.

Analytical Method

Cell growth was analyzed by measuring absorbance at ₅₀₀ nm (OD ₅₀₀ ). Further microscopic cell counts were determined in the counting chamber (0.02 mm depth) and dry cell weight (DCW) was determined (see Daich et al., J. Biotechnol. 64, 197-210 (1998)). The number of colony forming units (cfu) was determined by outcropping samples diluted on nutrient agar plates incubated for at least 3 days. Plasmid stability was then determined by overstamping these plates onto selective agar by replica plating techniques. The relationship between DCW, OD ₅₀₀ and cell counts was characterized as follows: 1 g / l of DCW corresponds to an OD ₅₀₀ of 4.5 ± 0.1 and a cell count of 1.8 × 10 ⁹ / ml. Glucose concentrations were determined using a commercial enzyme kit.

α-glucosidase concentration was determined after separation of the whole cell sample on an SDS gel (5% collection gel, 7% separation gel). Expression was performed by scanning the product strips and quantifying in comparison to standard products located on the gel at various concentrations.

result

this. E. coli RB791 pKK177glucC and E. coli. E. coli RB791 pKK177glucC pUBS520 was incubated in a stirred reactor using a glucose-limited batch feed method. After the first batch phase, constant feeding was started, and 3 hours after the start of feeding, expression of the α-glucosidase gene was induced by the addition of 1 mM IPTG. After induction, the α-glucosidase concentration increased, at which time the specific concentration of enzyme per cell reached its maximum value about 5 hours after induction and began to decrease in longer cultures (see FIG. 1C). The decrease in the specific concentration of α-glucosidase is due to overgrowth of the culture by plasmid-free cells. They have significant growth advantages after induction because the production of α-glucosidase adversely affects growth and also inhibits glucose uptake in the resulting cells. This accumulates glucose in the culture medium. Cells present in cultures that do not contain product genes are not affected by inducer IPTG but rather continue to grow without being limited by the high availability of glucose.

If the glucose solution is not added continuously but rather in short-cycle waves at about 1 minute intervals (see "Materials and Methods"), α-glucosidase will accumulate similar to a constant feed after induction. However, overgrowth of culture with plasmid-free cell populations can be prevented depending on the wave duration (see FIG. 1D). This positive effect on the inhibition of plasmid-free cells is not only in the strongly expressing system shown in FIG. It is also evident in the weak expression of α-glucosidase in the E. coli RB791 pKK177glucC system (see FIG. 2, Table 1). Moreover, the wave feeding has a slightly positive effect on the rate of synthesis in both cases after induction, in the first case also affecting the stability of the product in which more than 90% is present in inclusion body form. The definition of cycle time is an important factor. In the two examples shown, the extended cycle time of up to 4 minutes reduces the amount of product and thus yield (see FIGS. 1 and 2 and Table 1). In this case, overgrowth of the culture by plasmid-free cells is also reduced, but longer cycle times reduce the synthesis of the product or increase disruption.

E. with or without PUBS520. Production and Overgrowth by Plasmid-Free Cells in a Glucose-Limited Batch-Feed Culture of E. coli RB791 pKK177glucC Type of substrate added during batch-feed fermentation α-glucosidase yield [mg / g biomass] Plasmid-free cells [% of total population] 3 hours after induction 20 hours after induction 3 hours after induction 20 hours after induction RB791 pKK177glucC pUBS520 Constant supply 37 30 2 72 1 minute cycle 38 24 One 16 4 minute cycle 37 6 2.5 60 RB791 pKK177glucC 1 minute cycle 10 9 10 10 4 minute cycle 6 4.6 15 6.7

Claims (9)

Characterized by vibrating decreasing or increasing the concentration of the carbon / energy source in the culture at short intervals, A method for enhancing the yield of recombinant protein in a microbial fermentation process. The method of claim 1, Vibration is generated by varying the rate of administration of a feed solution containing a carbon / energy source. The method according to claim 1 or 2, Wherein the maximum duration of one cycle is four minutes and the duration of a single phase of such cycle is up to two minutes. The method according to any one of claims 1 to 3, Wherein the period of one cycle is one minute and the phase of this cycle is at most 75% of the total cycle time. The method according to any one of claims 1 to 3, Adding a carbon / energy source to the culture in a manner that periodically changes the rate of addition of the substrate solution only during certain portions of the process. The method according to any one of claims 1 to 5, Controlling the rate of administration by periodically activating or inactivating the addition of the feed solution. The method according to any one of claims 1 to 5, Wherein glucose, glycerol, lactose, galactose, methanol, acetate, molasses or starch are used as carbon / energy substrates. The method according to any one of claims 1 to 7, Depending on the promoter used, IPTG or indolyl acrylic acid (IAA), or lactose, arabinose, galactose or methanol (if not already used as an energy source) is added to the culture to induce the formation of recombinant products. How to. The method according to any one of claims 1 to 9, Varying the temperature when inducing the formation of the recombinant product.