Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter

Abstract

ABSTRACT To develop a functional phosphate-regulated promoter in Pichia pastoris , a phosphate-responsive gene, PHO89 , which encodes a putative sodium (Na + )-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter ( P PHO89 ) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P PHO89 was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1α and the glyceraldehyde-3-phosphate dehydrogenase promoter, P PHO89 exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P PHO89 -based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P PHO89 for controlled production of recombinant proteins in P. pastoris .