INDUSTRIAL FERMENTATION PROCESS FOR BACILLUS USING TEMPERATURE SHIFT

Abstract

The present invention relates to the field of industrial fermentation. In particular, it relates to a method for cultivating 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, (b) 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 cultivation during the first cultivation phase is carried out at a first temperature, and (c) cultivating for a second cultivation phase the Bacillus host cell culture obtained in step (b) 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 the cultivation during the second cultivation phase is carried out at a second temperature, said second temperature being higher than the first temperature. The invention also provides for a Bacillus host cell culture obtainable by the said method.

Claims

1. A method for cultivating 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; (b) 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 cultivation during the first cultivation phase is carried out at a first temperature; and (c) cultivating for a second cultivation phase the Bacillus host cell culture obtained in step (b) 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 the cultivation during the second cultivation phase is carried out at a second temperature, said second temperature being higher than the first temperature.

2. The method of claim 1, wherein said method further comprises obtaining the protein of interest from the Bacillus host cell culture obtained after step (c).

3. The method of claim 1, wherein the protein of interest is an enzyme.

4. The method of claim 1, wherein the expression construct comprises a nucleic acid sequence encoding the protein of interest operably linked to a promoter.

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

6. The method of claim 1, wherein during the first cultivation phase the at least one feed solution provides a carbon source at increasing rates.

7. The method of claim 1, wherein said second cultivation phase is carried out for a time of at least about 3 h up to about 96 h.

8. The method of claim 1, wherein during the second cultivation phase the 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 (b), wherein said constant rate or the starting rate of said decreasing rates or the staring rate of said rates increasing less than the rates in step (b) is below the maximum rate of the first cultivation phase.

9. The method of claim 1, wherein said first and said second temperature differ by about 3° C. to about 7°C.

10. The method of claim 1, wherein said first temperature is within the range of about 28° C. to about 32 °C.

11. The method of claim 1, wherein said second temperature is within the range of about 33° C. to about 37 °C.

12. The method of claim 1, wherein the yield of the protein of interest obtained after step c) is significantly increased compared to a control which has been obtained by carrying out the method wherein the said first and second temperature are identical.

13. The method of claim 12, wherein said yield is increased by at least 40%, at least 60%, at least 80%, at least 100%, at least 200%, at least 300% or at least 400%.

14. The method of claim 1, wherein said Bacillus is selected from the group consisting of: Bacillus licheniformis, Bacillus subtilis, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus jautus, Bacillus lentus, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus thuringiensis, and Bacillus velezensis.

15. The method of claim 1, wherein said expression construct for a gene encoding a protein of interest has been introduced into the Bacillus host cell by genetic modification.

16. The method of claim 1, wherein said at least one feed solution comprises at least one carbon source.

17. A Bacillus host cell culture obtainable by the method of claim 1.

Description

FIGURES

[0134] FIG. 1: Relative yields of amylases from fed-batch fermentations of Bacillus licheniformis at constant temperatures of 30° C. and 35° C. versus shifting temperature during fermentation from 30° C. to 35° C. Shown are two exemplified fed-batch fermentations Amylase 1 (A) and Amylase 2 (B).

[0135] FIG. 2: Relative enzyme yields from fed-batch fermentations at constant temperature and using temperature shift. (A) Relative yields of amylase 1 from fed-batch fermentations of Bacillus subtilis at constant temperatures of 30° C. versus shifting temperature during fermentation from 30° C. to 35° C. (B) Relative yields of mannanase from fed-batch fermentations of Bacillus licheniformis at constant temperatures of 30° C. versus shifting temperature during fermentation from 30° C. to 35° C.

[0136] FIG. 3: Optimizing time point of temperature shift from 30° C. to 35° C. by combining temperature shift with the reduction in the specific substrate uptake rate qs. (A) shows the glucose feed rate over the feed time. The total feed time was 70 h (corresponding to 100 %). (B) depicts the glucose feed rate over the relative amount of glucose added. (C) depicts the specific glucose uptake rate (qs) over the relative amount of glucose added. (D) depicts the amylase yield depending on the amount of total glucose added before the temperature shift. The arrow indicates the bar representing the combination of temperature shift and shift in feed rate.

EXAMPLES

[0137] 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 1: Shifting Temperature During Fermentation Increases Amylase Production in Bacillus Licheniformis

[0138] Unless otherwise stated the following experiments have been performed by applying standard equipment, methods, chemicals, and biochemicals as used in genetic engineering and fermentative production of chemical compounds by cultivation of microorganisms. See also Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold 20 Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and Chmiel et al. (Bioprocesstechnik 1. Einführung in die Bioverfahrenstechnik, Gustav Fischer Verlag, Stuttgart, 1991).

[0139] Alpha-amylase activity was determined by a method employing the substrate Ethyliden-4-nitrophenyl-α-D-maltoheptaoside (EPS). D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase. Following the cleavage an alpha-glucosidase liberates a PNP molecule which has a yellow color and thus can be measured by visible spectophotometry at 405 nm. Kits containing EPS substrate and alpha-glucosidase are available from Roche Costum Biotech (cat. No. 10880078t3) and are described in Lorentz K. et al. (2000), Clin. Chem., 46/5: 644 - 649. The slope of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alpha-amylase in question under the given set of conditions.

[0140] Bacillus licheniformis strains expressing amylase 1 or amylase 2 were cultivated in a fermentation process using a chemically defined fermentation medium providing the components listed in Table 1 and Table 2.

TABLE-US-00001 Macroelements provided in the fermentation process Compound Formula Added per initial mass [g/kg] Citric acid Monohydrate C.sub.6H.sub.8O.sub.7 * H.sub.2O 11.2 Calcium Ca 0.3 Sodium Na 1.6 Potassium P 4.0 Magnesium Mg 0.4 Sulfate SO.sub.4 2.9 Ammonium NH.sub.4 0.3 Phosphate PO.sub.4 15.8

TABLE-US-00002 Trace elements provided in the fermentation process Compound Symbol Added per initial mass [.Math.mol/kg] Manganese Mn 240 Zinc Zn 175 Copper Cu 320 Cobalt Co 11 Nickel Ni 3 Molybdenum Mo 20 Iron Fe 385

[0141] 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.

[0142] The feed was started upon depletion of the initial amount of 8 g/l glucose indicated by an increase of culture pH and glucose was added until > 200 g of glucose per kg initial fermentation volume were added to the bioreactor. The glucose feeding strategy consisted of an initial exponential feed phase with an exponential factor of 0.13 h.sup.-1 and a starting value of 1 g of glucose per L initial volume and hour where 28% of the total glucose were added to the bioreactor. This was followed by a second phase of constant glucose feeding with a rate corresponding to 35% of the maximum glucose feeding rate. In this second phase the rest of the glucose (72% of the total glucose) was added. pH was kept over 7.0 by addition of NH.sub.4OH.

[0143] The cultivation temperature was kept constant at either 30° C. or 35° C., resulting in relative amylase yields of 100% and 229% for amylase 1 and 100% and 143% for amylase 2, respectively. Starting the fermentation at a lower temperature of 30° C. and then increasing the temperature to 35° C. after the end of the exponential feeding phase increased the yield to 451% and 723% for amylase 1 and amylase 2, respectively. Thus, performing a shift in temperature during the fermentation from a lower temperature to a higher temperature increased productivity significantly compared to fermentations where temperature was kept constant at either the lower (30° C.) or higher (35° C.) temperature. Results are depicted in FIG. 1.

Example 2: Shifting Temperature During Fermentation Increases Amylase Production in Bacillus Subtilis

[0144] Enzyme activity was determined as described in Example 1. A Bacillus subtilis strain expressing amylase 1 was grown in mineral salt media in a fed-batch fermentation with glucose as carbon source as described in Example 1.

[0145] The cultivation temperature was kept constant at either 30° C. or the fermentation was started at 30° C. and then the temperature increased to 35° C. after the end of the exponential feeding phase. Performing a shift in temperature during the fermentation from a lower to a higher setpoint increased productivity significantly (49% increase) compared to fermentations where temperature was kept constant at 30° C. Results are shown in FIG. 2 (A).

Example 3: Shifting Temperature During Fermentation Increases Mannanase Production in BacilLus Licheniformis

[0146] A mannanase molecule as described in WO2021/058453 (Seq ID No:1) was expressed in Bacillus licheniformis. The Bacillus licheniformis strain was then grown in mineral salt media in a fedbatch fermentation with glucose as carbon source as described in Example 1. The cultivation temperature was kept constant at either 30° C. or the fermentation was started at 30° C. and then the temperature increased to 35° C. after the end of the exponential feeding phase. Mannanase titers were determined from cultivation samples over the course of the fermentations by CE-SDS electrophoresis according to standard test procedures known to a person skilled in the art. Performing a shift in temperature during the fermentation from a lower to a higher setpoint increased productivity significantly (33% increase) compared to fermentations where temperature was kept constant at 30° C. Results are shown in FIG. 2 (B).

Example 4: Combining Temperature Shift With Reduction of Specific Substrate Uptake Rate Q.SUB.s increases amylase yield

[0147] Enzyme activity was determined as described in Example 1. A Bacillus licheniformis strain expressing amylase 1 was grown in mineral salt media in a fed-batch fermentation with glucose as carbon source as described in Example 1.

[0148] After start of the glucose feeding, the shift in temperature from 30° C. to 35° C. was performed after different amounts glucose were added (0% = start of feeding). After addition of 28% of the total amount of glucose, the feed profile was shifted from an exponential profile to a constant feed, resulting in a reduction of the specific substrate uptake rate q.sub.s [gram glucose per gram cells and hour] to 35% of the maximum observed during the cultivation.

[0149] The maximum amylase yield was achieved by shifting the temperature in parallel with the switch to the constant feed rate (28% of glucose added of total amount of glucose added during the fermentation process) i.e. the reduction in the specific substrate uptake rate to 35% of its maximum. Performing the temperature shift before or after the reduction of q.sub.s resulted in lower product titers. Consequently, a synergetic effect was achieved by shifting cultivation temperature and q.sub.s at the same time. Results are shown in FIG. 3.