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 (PPHO89) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. PPHO89 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, PPHO89 exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple PPHO89-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of PPHO89 for controlled production of recombinant proteins in P. pastoris.
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