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 1alpha 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.

FIG. 1.

Expression vectors used in this study. CLLip, gene encoding lipase from Bacillus stearothermophilus fused to a CBD and linker of endoglucanase II from T. harzianum; AMss, α-amylase signal sequence from A. oryzae; Ampr, ampicillin resistance gene; P_PHO89, PHO89 promoter of P. pastoris sodium-coupled phosphate symporter; pBR322 ori, E. coli replication origin; HIS4, histidinol dehydrogenase gene from P. pastoris; TT, transcription termination and polyadenylation signal from the AOX1 gene; 3′AOX1, 3′ AOX1 downstream sequence AvrII, BglII, StuI, SalI, and NotI, restriction enzyme sites.

FIG. 2.

Nucleotide sequence of the P. pastoris-derived PHO89 gene with 5′ and 3′ flanking regions. The TATA box that is discussed in the text is enclosed in a box. The potential consensus sites (CACGTG/T) recognized by Pho4p are underlined, as is a stretch of seven amino acid residues (G-A-N-D-V-A-N) that is highly conserved in other sodium-coupled phosphate transporters.

FIG. 3.

Results obtained with batch cultures of P. pastoris GS115/pNPS-AM-CLLip with different initial phosphate concentrations. Batch cultures A (squares), B (triangles), and C (circles) contained initial concentrations of NaH₂PO₄·2H₂O of 2.5, 0.25, and 0.025 g/liter (as the sole phosphate source), respectively.

FIG. 4.

Western blot for batch cultures of P. pastoris GS115/pNPS-AM-CLLip described in the legend to Fig. 3. Lane M contained the standard marker. The numbers above the lanes indicate the culture times (in hours). The arrow indicates the position of the expressed CLLip.

FIG. 5.

Results obtained with batch cultures of P. pastoris GS115/pNPS-AM-CLLip with different initial concentrations of glucose. Circles, triangles, squares, and diamonds indicate batch cultures with initial glucose concentrations of 50, 110, 200 and 320 g/liter, respectively.

FIG. 6.

Results obtained with a fed-batch culture of P. pastoris GS115/pNPS-AM-CLLip with intermittent addition of glucose in complex medium.