Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica |
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Authors: | Mayson Brian E Kilburn Douglas G Zamost Bruce L Raymond Christopher K Lesnicki Gary J |
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Institution: | The Biotechnology Laboratory, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada. |
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Abstract: | The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter. Methanol concentrations during the induction phase directly affect cellular growth and protein yield. Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P. methanolica expressing the human transferrin N-lobe protein. The PMAD18 P. methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype. Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration. Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time. |
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Keywords: | Pichia methanolica gene expression system methanol monitoring and control methylotrophic yeast fermentation heterologous protein production |
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