A STRONG NITROGEN SOURCE-REGULATED PROMOTER FOR CONTROLLED EXPRESSIONOF FOREIGN GENES IN THE YEAST PICHIA-PASTORIS

In methylotrophic yeasts, glutathione-dependent formaldehyde dehydroge nase (FLD) is a key enzyme required for the metabolism of methanol as a carbon source and certain alkylated amines such as methylamine as ni trogen sources. We describe the isolation and characterization of the FLD1 gene from the yeast Pichia pastoris. The gene contains a single s hort intron with typical yeast-splicing signals near its 5' end, the f irst intron to be demonstrated in this yeast. The predicted FLD1 produ ct (Fld1p) is a protein of 379 amino acids (approx. 40 kDa) with 71% i dentity to the FLD protein sequence from the n-alkane-assimilating yea st Candida maltosa and 61-65% identity with dehydrogenase class III en zymes from humans and other higher eukaryotes. Using p-lactamase as a reporter, we show that the FLD1 promoter (P-FLD1) is strongly and inde pendently induced by either methanol as sole carbon source (with ammon ium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source). Furthermore, with either methanol or methylamine induction, levels of p-lactamase produced under control o f P-FLD1 are comparable to those obtained with the commonly used alcoh ol oxidase I gene promoter (P-AOX1). Thus, P-FLD1 is an attractive alt ernative to P-AOX1 for expression of foreign genes in P. pastoris, all owing the investigator a choice of carbon (methanol) or nitrogen sourc e (methylamine) regulation with the same expression strain. (C) 1998 E lsevier Science B.V. All rights reserved.