Vectors for gene expression and amplification in the yeast Yarrowia lipolytica

New vector systems were developed for gene expression in Y. lipolytica . Th ese plasmids contain: (a) as integration target sequences, either a rDNA re gion or the long terminal repeat zeta of the Y, lipolytica retrotransposon Ylr1; (b) the YlURA3 gene as selection marker for Y. lipolytica , either as the non-defective ura3d1 allele for single integration or the promotor tru ncated ura3d4 allele for multiple integration; (c) the inducible ICL1 or XP R2 promoters for gene expression; and (d) unique restriction sites for gene insertion, Multiple plasmid integration occurred as inserted tandem-repeat s, which are present at 3-39 copies per cell, A correlation between gene co py number and the expressed enzyme activity was demonstrated with Escherich ia coli lacZ as reporter gene under the control of the regulated ICL1 promo ter, Increases in copy numbers from 5 to 13 for the lacZ expression cassett es resulted in an up to 10-11-fold linear increase of the P-galactosidase a ctivity in multicopy transformants during their growth on ethanol or glucos e, compared with the low-copy replicative plasmid transformants (1.6 plasmi d copies), These new tools will enhance the interest in Y, lipolytica as an alternative host for heterologous protein production, Copyright (C) 2000 J ohn Wiley & Sons, Ltd.