Heterologous Protein Secretion Directed by a Repressible Acid Phosphatase System of Kluyveromyces lactis : Characterization of Upstream Region-Activating Sequences in the KIPHO5 Gene

Abstract

ABSTRACT Transcription of the repressible acid phosphatase gene ( KIPHO5 ) in Kluyveromyces lactis is strongly regulated in response to the level of inorganic phosphate (P i ) present in the growth medium. We have begun a study of the promoter region of this gene in order to identify sequences involved in the phosphate control of KIPHO5 expression and to design new expression-secretion systems in K. lactis . Deletion analysis and directed mutagenesis revealed two major identical upstream activating sequences (UAS) CACGTG at positions −430 (UAS1) and −192 (UAS2) relative to the ATG initiation codon. These sequences are identical to those described for Saccharomyces cerevisiae for the binding of Pho4p. Deletion or directed mutagenesis of either one or both UAS reduce KIPHO5 expression by the same amount (approximately 80%). When fused to the coding region of trout growth hormone cDNA ( tGH-II ), the promoter and signal peptide-encoding region of the phosphate-repressible KIPHO5 gene drives the expression of this gene and the secretion of the tGHII protein. Synthesis of tGHIIp in K. lactis transformants carrying this construct was found to be regulated by the P i present in the medium; derepression of heterologous protein expression can therefore be achieved by lowering the P i concentration.