Industrial fermentation process for bacillus using partial harvest

Abstract

The present invention relates to the field of industrial fermentation. In particular, it relates to method for producing a protein of interest from a Bacillus host cell comprising the steps of inoculating a fermentation medium with a Bacillus host cell comprising an expression construct for a gene encoding a protein of interest, cultivating the Bacillus host cell in said fermentation medium under conditions conducive for the growth of the Bacillus host cell and the expression of the protein of interest for a cultivation phase, wherein the cultivation of the Bacillus host cell during said cultivation phase comprises the addition of at least one feed solution and wherein the at least one feed solution provides a carbon source, and separating portions from said Bacillus host cell culture during the cultivation phase at different time points and obtaining the protein of interest from said portions.

Claims

1. A method for producing a protein of interest from a Bacillus host cell comprising the steps of: (a) inoculating a fermentation medium with a Bacillus host cell comprising an expression construct for a gene encoding a protein of interest; (b1) cultivating for a first cultivation phase the Bacillus host cell in said fermentation medium under conditions conducive for the growth of the Bacillus host cell and the expression of the protein of interest, wherein the cultivation of the Bacillus host cell comprises the addition of at least one feed solution and wherein the at least one feed solution provides a carbon source at increasing rates; (b2) cultivating for a second cultivation phase the Bacillus host cell culture obtained in step (b1) under conditions conducive for the growth of the Bacillus host cell and the expression of the protein of interest, wherein the cultivation comprises the addition of at least one feed solution and wherein at least one feed solution provides a carbon source at a constant rate, at decreasing rates, or at rates increasing less than the rates in step (b1), wherein said constant rate or the starting rate of said decreasing rates or the starting rate of said rates increasing less than the rates in step (b1) is below the maximum rate of the first cultivation phase; and (c) separating portions from said Bacillus host cell culture during the cultivation phase at different time points and obtaining the protein of interest from said portions.

2. The method of claim 1, wherein said increasing rates in step (b1) are exponentially increasing rates.

3. The method of claim 2, wherein during the first cultivation phase the at least one feed solution provides a carbon source at exponentially increasing rates with an exponential factor of at least 0.13 h.sup.1 and a starting amount of at least 1 g per liter and hour of the at least one carbon source.

4. The method of claim 1, wherein during said first cultivation phase a total amount of at least 50 g of said at least one carbon source per kg Bacillus host cell culture being initially present in step b) is added.

5. The method of claim 1, wherein said first cultivation phase is carried out for a time of at least 3 h up to 48 h.

6. The method of claim 1, wherein said at least one feed solution in step (b2) provides the said carbon source at a constant rate.

7. The method of claim 6, wherein said constant rate is within the range of 70% to 20% of the maximum feeding rate for the at least one carbon source applied in the first cultivation phase.

8. The method of claim 1, wherein said second cultivation phase is carried out for a time of at least 40 h up to 120 h.

9. The method of claim 1, wherein said cultivation phase in step (c) during which said separating of portions from said Bacillus host cell culture at different time points is carried out is the second cultivation phase.

10. The method of claim 1, wherein cultivation during the first cultivation phase is carried out at a first temperature and the cultivation during the second cultivation phase is carried out at a second temperature, said second temperature being higher than the first temperature.

11. The method of claim 1, wherein, after inoculation of the fermentation medium and prior to the first cultivation phase, the method further comprises depleting at least one carbon source.

12. The method of claim 1, wherein the first time point of said different time points is 32 h after onset of the cultivation phase.

13. The method of claim 1, wherein the time difference between said different time points is between 6 h to 10 h.

Description

FIGURES

(1) FIG. 1: Glucose feed rates relative to initial reactor volume for fermentations with either 5.6 g of glucose per L initial volume and without partial harvest (grey dotted line) or 8.8 g of glucose per L initial volume in linear feed phase with partial harvest (black solid line). The arrow indicates the start of feeding at 10 hours after process start.

(2) FIG. 2: Time course of mass of harvest per maximum occupied reactor volume (RV) for fermentations without (grey dotted line) and with (black solid line) partial harvest.

(3) FIG. 3: Time course of normalized yield on glucose for fermentations without (squares) and with (diamonds) partial harvest.

EXAMPLES

(4) The invention will now be illustrated by working Examples. Theses working Examples must not construed, whatsoever, as limitations of the scope of the invention.

Example: Stabilizing Yield of Alkaline Protease 1 on Glucose by Partial Harvest Strategy

(5) Bacillus licheniformis strains expressing alkaline protease 1 were cultivated in a fermentation process using a chemically defined fermentation medium providing the components listed in Table 1.

(6) TABLE-US-00001 TABLE 1 Macroelements provided in the fermentation process Concentration [g/L initial volume] with partial Compound Formula without harvest Citric acid monohydrate C.sub.6H.sub.8O.sub.7 14.3 18.8 Calcium sulfate dihydrate CaSO.sub.4*2H.sub.2O 1.7 2.1 Monopotassium phosphate KH.sub.2PO.sub.4 20.5 42.1 Disodium phosphate Na.sub.2HPO.sub.4 5.3 5.3 Magnesium sulfate heptahydrate MgSO.sub.4*7H.sub.2O 3.6 4.7 Ammonia NH.sub.3 26 33

(7) TABLE-US-00002 TABLE 2 Microelements provided in the fermentation process Concentration [M initial volume] with partial Compound Formula without harvest Manganese Mn 361 499 Zinc Zn 266 366 Copper Cu 498 675 Cobalt Co 16 22 Nickel Ni 29 40 Molybdenum Mb 4 6 Iron Fe 584 806

(8) The fermentation was started with a medium containing 8 g/l glucose. A solution containing 50% glucose was used as feed solution. The pH was adjusted during fermentation using ammonia.

(9) The feed was started 10 hours after process start upon depletion of the initial amount of 8 g/l glucose indicated by an increase of culture pH by 0.2 pH units. The glucose feeding strategy consisted of an exponential feed phase with an exponential factor of 0.13 h1 and a starting value of 1 g of glucose per L initial volume and hour for 22 h. This was followed by a phase of constant glucose feeding with a rate of either 5.6 g or 8.8 g of glucose per L initial volume and hour for 68 h (FIG. 1). In case of the latter a volume of 100 mL per L initial volume was taken as partial harvest every 8 h from 32 h of fermentation time on, whereas in case of the former only minor sampling volumes for analytics were taken (FIG. 2). In both fermentations pH was kept over 7.0 by addition of NH.sub.4OH. The cultivation temperature was kept constant at 30 C.

(10) The fermentation without partial harvest reaches a normalized maximum product yield on glucose of 1.00 around 45 h after start of fermentation (FIG. 3). Thereafter, the yield decreases in a steady fashion to only 0.30 normalized product yield on glucose after 85 h of process time, thus declining in yield by 70% in 40 h.

(11) The fermentation with partial harvest reaches a normalized product yield on glucose of 0.60 around 47 h and reaches its maximum yield of 0.62 at 55 h after start of fermentation (FIG. 3). After 87 h of fermentation, the normalized yield on glucose is still at 0.41, thus declining in yield by 34% in 40 h. This indicates clearly a stabilizing effect of the partial harvest strategy on the product yield on glucose.