Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes
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Increased copy number of foreign gene can result in the alteration of normal metabolism in Pichia pastoris. To better understand the effect of foreign gene dosage on the cellular physiology of P. pastoris cells, comparative transcriptional analysis was performed among three P. pastoris strains carrying 0, 6, and 18 copies of porcine insulin precursor (PIP) expression cassettes, respectively. mRNA levels of 13 selected genes involved in methanol metabolic pathway, central metabolic pathway, protein folding, and oxidative stress were determined by real-time PCR. Results showed that enhanced PIP copy number resulted in an increase in PIP mRNA and also in folding stress on the yeast cells’ endoplasmic reticulum. The metabolism of 6-copy P. pastoris strain was not significantly changed as compared to 0-copy strain (control). In contrast, physiology of 18-copy strain was remarkably affected, characterized by the upregulation of antioxidative genes and readjusted expression level of methanol metabolic pathway genes. These data suggested that high copy P. pastoris strain might be suffering from protein folding-related oxidative stress and insufficient supply of carbon and energy sources.
KeywordsGene dosage Pichia pastoris Methanol metabolism Cell physiology mRNA quantitative analysis
We thank Professor James M. Gregg (Keck Graduate Institute of Applied Life Sciences) for providing sequence information. This research was supported by a grant from National Natural Science Foundation of China (no. 30560006) and by National Basic Research Program of China (no. 2007CB714303).
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