Feeding of mixed-carbon-resource during the expression phase in cultivation of recombinant Pichia pastoris expressing angiostatin.
- Author:
Jing-Li XIE
1
;
Qing-Wei ZHOU
;
Li ZHANG
;
Qin YE
;
Li XIN
;
Peng DU
;
Ren-Bao GAN
Author Information
1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237.
- Publication Type:Journal Article
- MeSH:
Angiostatins;
genetics;
metabolism;
physiology;
Biotechnology;
methods;
Carbon;
metabolism;
Fermentation;
physiology;
Glycerol;
metabolism;
Methanol;
metabolism;
Oxygen;
metabolism;
Pichia;
genetics;
metabolism
- From:Chinese Journal of Biotechnology
2003;19(4):467-470
- CountryChina
- Language:Chinese
-
Abstract:
A recombinant strain of Pichia pastoris with a phenotype of Muts was used to produce angiostatin in a 5-L fermentor. The methanol utilization ability of the present strain was weak, which resulted in extremely low growth rate and angiostatin productivity during the expression phase with methanol as the sole carbon source. To enhance the cell density and angiostatin expression level, mixed-carbon-source of glycerol-methanol was used in the expression phase. The methanol concentration was well controlled at 5 g/L by a methanol sensor and control system, and glycerol was continuously fed into the fermentor to achieve a higher cell density. 120 g/L of cells and 39 mg/L of angiostatin were reached at the end of fermentation which lasted 110 h. The mean specific cell growth rate in the expression phase was 0.01 h(-1), and the mean specific angiostatin productivity was 0.006 mg/(g x h). According to the data obtained in several runs of fermentation in which glycerol was fed at different rates, a higher mean specific angiostatin productivity was reached at the mean specific cell growth rate of 0.012 h(-1). To avoid the repression of angiostatin expression caused by residual glycerol and ethanol accumulation due to overfeeding of glycerol, glycerol addition was controlled to produce continuous oscillations in dissolved oxygen, because the change of dissolved oxygen concentration could deliver the information of available carbon source in the fermentation broth. Controlled glycerol feeding also avoided the problem of oxygen limitation brought by high cell density, and thus decreased the cooling requirement of the fermentor. Cell density reached 150 g/L at the end of fermentation, and angiostatin level reached 108 mg/L after an expression period of 96 h when the mean specific growth rate was maintained at 0.012 h(-1) by using the glycerol feeding strategy to result in the oscillations in dissolved oxygen. The mean specific angiostatin productivity was improved to 0.02 mg/(g x h). The apparent cell yield on glycerol and methanol were respectively 0.69 g/g and 0.93 g/g, higher than those in the fermentation without using the feeding strategy with dissolved oxygen as the indicator of metabolism.
