Abstract
Several factors affect protein expression in Pichia pastoris, one among them is the carbon source. In this work, we studied the effect of this factor on the expression level of rabies virus glycoprotein (RABV‐G) in two recombinant clones harboring seven copies of the gene of interest. The expression was driven either by the constitutive glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase1 ( AOX1) promoter. Clones were compared in terms of cell physiology and carbon source metabolism. The transcription levels of 16 key genes involved in the central metabolic pathway, the methanol catabolism, and the oxidative stress were investigated in both clones. Cell size, as a parameter reflecting cell physiological changes, was also monitored. Our results showed that when glucose was used as the sole carbon source, large cells were obtained. Transcript levels of the genes of the central metabolic pathway were also upregulated, whereas antioxidative gene transcript levels were low. By contrast, the use of methanol as a carbon source generated small cells and a shift in carbon metabolism toward the dissimilatory pathway by the upregulation of formaldehyde dehydrogenase gene and the downregulation of those of the central metabolic. These observations are in favor of the use of glucose to enhance the expression of RABV‐G in P. pastoris.
Highlights
P. pastoris is an expression system widely used for protein production, its physiology during heterologous protein expression using different carbon sources has not been extensively studied as it was done for S. cerevisiae and E. coli
We studied the effect of carbon source on the physiology of recombinant clones of P. pastoris expressing rabies virus glycoprotein (RABV-G)
Cell size is considered as one of the most important determinant of cellular physiology; cell size of gap7 and aox7 clones grown on glucose or methanol, respectively, was determined
Summary
The importance of P. pastoris stems from the high cell density level that can be achieved during bioreactor culture and the low level of host cell proteins secreted into the culture medium, among others (Ahmad, Hirz, Pichler, & Schwab, 2014). Low production levels are still observed for some proteins. Heterologous protein expression in yeasts can be affected by different factors. In P. pastoris potential limiting factors of foreign protein expression are gene dosage
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