Journal
JOURNAL OF BIOTECHNOLOGY
Volume 131, Issue 1, Pages 57-66Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2007.05.019
Keywords
Pichia pastoris; avidin; mixed substrate; sorbitol; calorimetry; high cell density
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The advantages of mixed feeds of sorbitol and methanol for the production of recombinant proteins with Pichia pastoris were analyzed quantitatively. The influence of the methanol-sorbitol ratio in the feed medium was investigated on growth stoichiometry and recombinant protein productivity with a P pastoris Mut(+) strain secreting avidin by performing a transient nutrient gradient in continuous cultivation at a dilution rate of 0.03 h(-1). Results showed that mixed feeds of sorbitol and methanol instead of methanol as sole carbon source can improve the productivity in recombinant avidin due to increased biomass yields during mixed substrate growth. The highest volumetric avidin productivity was achieved at a methanol fraction of 43% C-mol C-mol(-1) in the feed medium: the volumetric avidin productivity was 1.3-fold higher than during chemostat culture on methanol. The heat production and the oxygen consumption rates were reduced by about 38% for a given dry cell weight concentration at this methanol fraction, features which are very useful for high cell density cultures. Results were in good agreement with a high cell density fed-batch culture performed with a mixed feed of 43% methanol and 57% sorbitol C-mol(-1) at a specific growth rate of 0.03 h(-1) during the induction phase. Moreover, it was confirmed that sorbitol accumulation in the culture medium does not affect recombinant avidin productivity, which can especially be advantageous during large-scale cultures where transient substrate accumulation can result from imperfect mixing. (c) 2007 Elsevier B.V. All rights reserved.
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