Studies on the alcohol oxidase (AOX1) promoter of Pichia pastoris
Pichia pastoris is an outstanding host for high-level heterologous gene expression, but there is still much interest in improving the productivity of recombinant protein production. Ethanol repression of the AOX1 promoter was investigated by using GS115 (Mut+) and MC100-3 (mut−) strains expressing intracellular AOX1::lacZ fusions. The addition of 10 mg L−1 ethanol at the start of methanol induction, delayed β-galactosidase production and methanol utilization for four hours in shake flask experiments. When ethanol and acetate were added together, all of the ethanol was converted to acetate, which also represses the AOX1 promoter.
The growth of P. pastoris in a mixture of either glycerol or glucose and methanol follows a diauxic growth, with C1 utilizing enzymes being repressed. Therefore, none of these carbon sources can be used as a mixture with methanol especially in shake flask culture of AOX-deficient P. pastoris. Among the tested carbon sources alanine, sorbitol, mannitol and trehalose did not repress AOX activity when methanol was used as an inducer in mut− strain of P. pastoris. A mut − strain growing in media containing trehalose, alanine, sorbitol and mannitol with methanol (0.5%) as an inducing agent expressed as much or higher β-galactosidase as compared to the mut+ growing in methanol containing media.
The regulation of the AOX1 promoter of P. pastoris was investigated by systematic deletions and DNA-protein interactions. Using the sequential deletion analysis, the 1120 by 5′ upstream region of AOX1 gene was divided into six subfragments. Each fragment had a significant effect on the promoter activity, however, the deletion analysis did not allow the exact localization of the upstream functions. Nevertheless, deletion of the fragment E (−273/−123) resulted in 84% reduction in native promoter activity. The fragments A (−960/−688) and C (−539/−399) contain sequences for DNA binding proteins. We show evidence that these proteins are different since these two fragments do not compete for the same protein by gel shift assay, and their nature remains to be characterized.
0307: Molecular biology