Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter

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 1␣ 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.Pichia pastoris is a methylotrophic yeast species that is increasingly used as a host system for heterologous protein expression for both academic and industrial purposes. To date, more than 500 proteins have been cloned and expressed using this system (19; http://faculty.kgi.edu/cregg/index htm), and it has provided the protein production platform for several structural genomics programs (25,35).In this system, most recombinant proteins have been produced using the alcohol oxidase I promoter (P AOX1 ), which is completely repressed when cells are grown on glucose and is induced in the presence of methanol (31). However, there are circumstances in which the promoter may not be suitable; the principal problem is that the highly volatile and inflammable compound methanol is required for transcription (29). Use of methanol for the induction of gene expression is often not permitted in the production of food products since methanol is a petroleum by-product (13, 33). Also, P AOX1 -based fermentation requires relatively long times and sophisticated feeding strategies for maintenance of the induction phase. These properties hamper the industrial applicability of this approach. Therefore, a promoter that does not require methanol is attractive for expression of foreign genes in P. pastoris. Potential promoters that are alternatives to P AOX1 include the glutathione-dependent formaldehyde dehydrogenase promoter (P FLD1 ) (26), for which either methanol or methylamine can act as an inducer, and the glyceraldehyde-3-phosphate dehydrogenase promoter (P GAP ) (33), which is a strong constitutive promoter in various microorganisms. Recently, we also developed the translation elongation factor 1␣ promoter (P TEF1 ), with more highly growth-associated expression characteristics, as an alternative to P GAP (2). The current study describes the use of a phosphate-responsive promoter as an alternative to constitutive and inducible promoters to drive expres...

Carbon source dependent promoters in yeasts

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Genetically modified microorganisms as producers of biologically active compounds

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Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production

“…Furthermore the use of the ethanol inducible P ICL1 (isocitrate lyase), (Menendez et al, 2003) or the promoter of the peroxisomal matrix protein Pex8 which can be induced by methanol or oleate (Liu et al, 1995) have been described but none of the latter promoters have been applied for routine production processes so far. Very recently, a phosphate responsible promoter of the putative sodium (Na + )-coupled phosphate symporter Pho89 was isolated which is highly active in phosphate limiting conditions (Ahn et al, 2009). …”