Fermentation process for increasing production level of recombinant human collagen

Abstract

The present invention provides a fermentation process for improving the production level of a recombinant human collagen. The process comprises inoculating a sterilized fermentation medium with a Pichia yeast solution, then performing fermentation culturing for 14-18 hrs, adding methanol to induce the expression, upon which sodium pyruvate is added to the fermentation medium, wherein the amount of the sodium pyruvate added is 0.01-10 g/L. In the fermentation process of the present invention, the sodium pyruvate is added in the methanol-induced expression stage, so that the biosynthesis rate of the recombinant human collagen is improved; and a continuous feed-batch mode is adopted, so that the biosynthesis rate of the recombinant human collagen is further improved, and the fermentation time is shortened. Meanwhile, the expression level of the recombinant human collagen is increased, the fermentation level is increased by more than 20%, and the production cost is reduced. The fermentation process is especially suitable for industrialized mass production of recombinant human collagen, and is of huge practical application value in industrial production.

Claims

1. A fermentation process for increasing the production level of a recombinant human collagen, comprising: inoculating a sterilized fermentation medium with a Pichia yeast solution; culturing the fermentation medium for 14-18 hours; adding methanol to the fermentation medium to provide a methanol-induced expression stage, wherein the Pichia yeast expresses the recombinant human collagen; and adding sodium pyruvate to the fermentation medium during the methanol-induced expression stage, wherein the amount of the sodium pyruvate added is 0.01-10 g/L, and wherein the Pichia yeast expresses the recombinant human collagen, wherein the recombinant human collagen is produced at higher levels than a process where sodium pyruvate is not added to the fermentation medium during the methanol-induced expression stage.

2. The fermentation process according to claim 1, wherein the amount of the sodium pyruvate added is 0.1-1 g/L.

3. The fermentation process according to claim 1, wherein the sodium pyruvate is added in a fed-batch mode.

4. The fermentation process according to claim 1, wherein the fermentation medium of the Pichia yeast has a composition comprising 6.6-26.7 mL/L of an 85% aqueous solution of H.sub.3PO.sub.4, 0.3-1.175 g/L of CaSO.sub.4.Math.2H.sub.2O, 4.5-18.2 g/L of K.sub.2SO.sub.4.Math.3.7-14.9 g/L of MgSO.sub.4.Math.7H.sub.2O, 1.0-4.13 g/L of KOH, 10.0-40.0 g/L of glycerol, and 0.435-4.35 mL/L of a PTM1 solution.

5. The fermentation process according to claim 1, wherein in the fermentation process, the Pichia yeast solution is inoculated in an amount of 8-12%, the fermentation temperature is 28-30° C., the pH is adjusted to 5.0-5.2 with aqueous ammonia, the dissolved oxygen is not less than 20%, and the induction time with methanol is 90-120 hrs.

6. The fermentation process according to claim 2, wherein the sodium pyruvate is added in a fed-batch mode.

Description

DETAILED DESCRIPTION

(1) In a specific example of the present invention, the fungal strain used is a Pichia yeast expressing a recombinant human collagen, which is Pichia pastoris deposited in China General Microbiological Culture Collection Center (CGMCC) under CGMCC Accession No. 5021 on Jun. 29, 2011.

(2) Refer to the operation manual of Invitrogen for the PTM1 solution mentioned in the present invention, which has a specific composition comprising 6.0 g/L of CuSO.sub.4.5H.sub.2O; 0.08 g/L of NaI; 3.0 g/L of MnSO.sub.4.H.sub.2O; 0.2 g/L of NaMoO.sub.4.2H.sub.2O; 0.02 g/L of H.sub.3BO.sub.3; 0.5 g/L of CoCl.sub.2; 20.0 g/L of ZnCl.sub.2; 65.0 g/L of FeSO.sub.4.7H.sub.2O; 0.2 g/L of biotin; and 5.0 mL/L of H.sub.2SO.sub.4, sterilized by filtration through a 0.22 μm filter membrane, and stored at room temperature.

(3) The present invention will be further described in detail below with reference to examples and accompanying drawings.

Example 1

(4) Fermentation medium: 85% H.sub.3PO.sub.4 26.7 mL/L; CaSO.sub.4.2H.sub.2O 1.175 g/L; K.sub.2SO.sub.4 18.2 g/L; MgSO.sub.4.7H.sub.2O 14.9 g/L; KOH 4.13 g/L; glycerol 40.0 g/L; and PTM1 4.35 mL/L.

(5) A seed culture was added to a 1000 L fermentor containing 500 L of a fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the fermentor pressure was 0.05 MPa, and the air flow rate and the stirring speed were adjusted so that the dissolved oxygen (DO) was >30%. When the carbon source was depleted, the dissolved oxygen rose sharply. 50% glycerol was added in a fed-batch mode, and the carbon source was supplemented. The addition of glycerol was stopped when the wet weight of the fungal cells was 212 g/L. At this time, the fermentation culturing time was 16 hrs. After the glycerol was depleted, methanol was added in a fed-batch mode, the process proceeded to a methanol-induced culture stage, and sodium pyruvate was added in one portion in an amount of 0.01 g/L of the fermentation medium. The rotation speed, the air flow rate and the fed-batch addition rate of methanol were adjusted to allow the dissolved oxygen (DO) to reach >20%. After 96 hrs of induced fermentation, the fermentation was completed. The fermentation liquid was collected, and the supernatant of the fermentation liquid was obtained by centrifugation. The final concentration of the recombinant human collagen is 16.0 g/L as detected by HPLC. The fermentation period is 112 hrs, and the fermentation production level is 0.143 g/L.Math.h, which is 23.3% higher than that of the control group.

Example 2

(6) Fermentation medium: 85% H.sub.3PO.sub.4 26.7 mL/L; CaSO.sub.4.2H.sub.2O 1.175 g/L; K.sub.2SO.sub.4 18.2 g/L; MgSO.sub.4.7H.sub.2O 14.9 g/L; KOH 4.13 g/L; glycerol 40.0 g/L; and PTM1 4.35 mL/L.

(7) A seed culture was added to a 1000 L fermentor containing 500 L of a fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the fermentor pressure was 0.05 MPa, and the air flow rate and the stirring speed were adjusted so that the dissolved oxygen (DO) was >30%. When the carbon source was depleted, the dissolved oxygen rose sharply. 50% glycerol was added in a fed-batch mode, and the carbon source was supplemented. The addition of glycerol was stopped when the wet weight of the fungal cells was 210 g/L. At this time, the fermentation culturing time was 16 hrs. After the glycerol was depleted, methanol was added in a fed-batch mode, the process proceeded to a methanol-induced culture stage, and sodium pyruvate was added in one portion in an amount of 10 g/L. The rotation speed, the air flow rate and the fed-batch addition rate of methanol were adjusted to allow the dissolved oxygen (DO) to reach >20%. After 96 hrs of induced fermentation, the fermentation was completed. The fermentation liquid was collected, and the supernatant of the fermentation liquid was obtained by centrifugation. The final concentration of the recombinant human collagen is 16.8 g/L as detected by HPLC. The fermentation period is 112 hrs, and the fermentation production level is 0.150 g/L.Math.h, which is 29.3% higher than that of the control group.

Example 3

(8) Fermentation medium: 85% H.sub.3PO.sub.4 26.7 mL/L; CaSO.sub.4.2H.sub.2O 1.175 g/L; K.sub.2SO.sub.4 18.2 g/L; MgSO.sub.4.7H.sub.2O 14.9 g/L; KOH 4.13 g/L; glycerol 40.0 g/L; and PTM1 4.35 mL/L.

(9) A seed culture was added to a 1000 L fermentor containing 500 L of a fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the fermentor pressure was 0.05 MPa, and the air flow rate and the stirring speed were adjusted so that the dissolved oxygen (DO) was >30%. When the carbon source was depleted, the dissolved oxygen rose sharply. 50% glycerol was added in a fed-batch mode, and the carbon source was supplemented. The addition of glycerol was stopped when the wet weight of the fungal cells was 216 g/L. At this time, the fermentation culturing time was 17 hrs. After the glycerol was depleted, methanol was added in a fed-batch mode, the process proceeded to a methanol-induced culture stage, and sodium pyruvate was added in one portion in an amount of 0.1 g/L. The rotation speed, the air flow rate and the fed-batch addition rate of methanol were adjusted to allow the dissolved oxygen (DO) to reach >20%. After 96 hrs of induced fermentation, the fermentation was completed. The fermentation liquid was collected, and the supernatant of the fermentation liquid was obtained by centrifugation. The final concentration of the recombinant human collagen is 17.8 g/L as detected by HPLC. The fermentation period is 113 hrs, and the fermentation production level is 0.158 g/L.Math.h, which is 36.2% higher than that of the control group.

Example 4

(10) Fermentation medium: 85% H.sub.3PO.sub.4 26.7 mL/L; CaSO.sub.4.2H.sub.2O 1.175 g/L; K.sub.2SO.sub.4 18.2 g/L; MgSO.sub.4.7H.sub.2O 14.9 g/L; KOH 4.13 g/L; glycerol 40.0 g/L; and PTM1 4.35 mL/L.

(11) A seed culture was added to a 1000 L fermentor containing 500 L of a fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the fermentor pressure was 0.05 MPa, and the air flow rate and the stirring speed were adjusted so that the dissolved oxygen (DO) was >30%. When the carbon source was depleted, the dissolved oxygen rose sharply. 50% glycerol was added in a fed-batch mode, and the carbon source was supplemented. The addition of glycerol was stopped when the wet weight of the fungal cells was 213 g/L. At this time, the fermentation culturing time was 18 hrs. After the glycerol was depleted, methanol was added in a fed-batch mode, the process proceeded to a methanol-induced culture stage, and a sodium pyruvate solution was added in a continuous fed-batch mode in a total amount of 0.1 g/L until the fermentation was completed. The rotation speed, the air flow rate and the fed-batch addition rate of methanol were adjusted to allow the dissolved oxygen (DO) to reach >20%. After 90 hrs of induced fermentation, the fermentation was completed. The fermentation liquid was collected, and the supernatant of the fermentation liquid was obtained by centrifugation. The final concentration of the recombinant human collagen is 18.2 g/L as detected by HPLC. The fermentation period is 108 hrs, and the fermentation production level is 0.169 g/L.Math.h, which is 45.3% higher than that of the control group.

Example 5

(12) Fermentation medium: 85% H.sub.3PO.sub.4 26.7 mL/L; CaSO.sub.4.2H.sub.2O 1.175 g/L; K.sub.2SO.sub.4 18.2 g/L; MgSO.sub.4.7H.sub.2O 14.9 g/L; KOH 4.13 g/L; glycerol 40.0 g/L; and PTM1 4.35 mL/L.

(13) A seed culture was added to a 1000 L fermentor containing 500 L of a fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the fermentor pressure was 0.05 MPa, and the air flow rate and the stirring speed were adjusted so that the dissolved oxygen (DO) was >30%. When the carbon source was depleted, the dissolved oxygen rose sharply. 50% glycerol was added in a fed-batch mode, and the carbon source was supplemented. The addition of glycerol was stopped when the wet weight of the fungal cells was 212 g/L. At this time, the fermentation culturing time was 16 hrs. After the glycerol was depleted, methanol was added in a fed-batch mode, the process proceeded to a methanol-induced culture stage, and a sodium pyruvate solution was added in a continuous fed-batch mode in a total amount of 1 g/L until the fermentation was completed. The rotation speed, the air flow rate and the fed-batch addition rate of methanol were adjusted to allow the dissolved oxygen (DO) to reach >20%. After 96 hrs of induced fermentation, the fermentation was completed. The fermentation liquid was collected, and the supernatant of the fermentation liquid was obtained by centrifugation. The final concentration of the recombinant human collagen is 18.0 g/L as detected by HPLC. The fermentation period is 112 hrs, and the fermentation production level is 0.161 g/L.Math.h, which is 38.8% higher than that of the control group.

Example 6

(14) Fermentation medium: 85% H.sub.3PO.sub.4 26.7 mL/L; CaSO.sub.4.2H.sub.2O 1.175 g/L; K.sub.2SO.sub.4 18.2 g/L; MgSO.sub.4.7H.sub.2O 14.9 g/L; KOH 4.13 g/L; glycerol 40.0 g/L; and PTM1 4.35 mL/L.

(15) A seed culture was added to a 1000 L fermentor containing 500 L of a fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the fermentor pressure was 0.05 MPa, and the air flow rate and the stirring speed were adjusted so that the dissolved oxygen (DO) was >30%. When the carbon source was depleted, the dissolved oxygen rose sharply. 50% glycerol was added in a fed-batch mode, and the carbon source was supplemented. The addition of glycerol was stopped when the wet weight of the fungal cells was 210 g/L. At this time, the fermentation culturing time was 17 hrs. After the glycerol was depleted, methanol was added in a fed-batch mode, the process proceeded to a methanol-induced culture stage, and sodium pyruvate was added in one portion in an amount of 1 g/L. The rotation speed, the air flow rate and the fed-batch addition rate of methanol were adjusted to allow the dissolved oxygen (DO) to reach >20%. After 96 hrs of induced fermentation, the fermentation was completed. The fermentation liquid was collected, and the supernatant of the fermentation liquid was obtained by centrifugation. The final concentration of the recombinant human collagen is 17.5 g/L as detected by HPLC. The fermentation period is 113 hrs, and the fermentation production level is 0.155 g/L.Math.h, which is 33.6% higher than that of the control group.

Example 7

(16) Fermentation medium: 85% H.sub.3PO.sub.4 6.6 mL/L; CaSO.sub.4.2H.sub.2O 0.3 g/L; K.sub.2SO.sub.4 4.5 g/L; MgSO.sub.4.7H.sub.2O 3.7 g/L; KOH 1 g/L; glycerol 10.0 g/L; and PTM1 0.435 mL/L.

(17) A seed culture was added to a 1000 L fermentor containing 500 L of a fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the fermentor pressure was 0.05 MPa, and the air flow rate and the stirring speed were adjusted so that the dissolved oxygen (DO) was >30%. When the carbon source was depleted, the dissolved oxygen rose sharply. 50% glycerol was added in a fed-batch mode, and the carbon source was supplemented. The addition of glycerol was stopped when the wet weight of the fungal cells was 210 g/L. At this time, the fermentation culturing time was 17 hrs. After the glycerol was depleted, methanol was added in a fed-batch mode, the process proceeded to a methanol-induced culture stage, and sodium pyruvate was added in one portion in an amount of 1 g/L. The rotation speed, the air flow rate and the fed-batch addition rate of methanol were adjusted to allow the dissolved oxygen (DO) to reach >20%. After 96 hrs of induced fermentation, the fermentation was completed. The fermentation liquid was collected, and the supernatant of the fermentation liquid was obtained by centrifugation. The final concentration of the recombinant human collagen is 17.1 g/L as detected by HPLC. The fermentation period is 113 hrs, and the fermentation production level is 0.151 g/L.Math.h, which is 30.2% higher than that of the control group.

Comparative Example

(18) The fermentation medium is a basal salt medium (BSM, see CN102443057B): 85% H3PO4 26.7 mL/L; CaSO4.2H2O 1.175 g/L; K2SO4 18.2 g/L; MgSO4.7H2O 14.9 g/L; KOH 4.13 g/L; glycerol 40.0 g/L; and PTM1 4.35 mL/L.

(19) A seed culture was added to a 1000 L fermentor containing 500 L of a fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the fermentor pressure was 0.05 MPa, and the air flow rate and the stirring speed were adjusted so that the dissolved oxygen (DO) was >30%. When the carbon source was depleted, the dissolved oxygen rose sharply. 50% glycerol was added in a fed-batch mode, and the carbon source was supplemented. The addition of glycerol was stopped when the wet weight of the fungal cells was 218 g/L. At this time, the fermentation culturing time was 16 hrs. After the glycerol was depleted, methanol was added in a fed-batch mode, and the process proceeded to a methanol-induced culture stage. The rotation speed, the air flow rate and the fed-batch addition rate of methanol were adjusted to allow the dissolved oxygen (DO) to reach >20%. After 120 hrs of induced fermentation, the fermentation was completed. The fermentation liquid was collected, and the supernatant of the fermentation liquid was obtained by centrifugation. The final concentration of the recombinant human collagen is 15.8 g/L as detected by HPLC. The fermentation period is 136 hrs, and the fermentation production level is 0.116 g/L.Math.h.