microbiology, Vol 145, 75-87, Copyright © 1999 by Society for General Microbiology

ARTICLES

Functional analysis of upstream regulating regions from the Yarrowia lipolytica XPR2 promoter

C Madzak, S Blanchin-Roland, RR Cordero Otero and C Gaillardin
Laboratoire de Genetique Moleculaire et Cellulaire INRA-CNRS, Centre de Biotechnologies Agro-Industrielles, Institut National Agronomique Paris- Grignon, Thiverval-Grignon, France. madzak@cardere.grignon.inra.fr

The XPR2 gene from Yarrowia lipolytica encodes an inducible alkaline extracellular protease. Its complex regulation involves pH, carbon, nitrogen and peptones. Two previously identified upstream activating sequence (UAS) regions were analysed in a reporter system, outside the XPR2 context. Fragments from the UAS regions were inserted upstream of a minimal LEU2 promoter directing the expression of a reporter gene. The activity of the hybrid promoters was assessed following integration into the Y. lipolytica genome. This study confirmed the presence of two UASs composed of several interacting elements. Within the distal UAS (UAS1), a TUF/RAP1 binding site exhibited a UAS activity, which was enhanced by the presence of two adjacent repeats, overlapping sites similar to the CAR1 upstream repressing sequence from Saccharomyces cerevisiae. Within the proximal UAS (UAS2), the UAS activity required the interaction of both an ABF1-like binding site and a decameric repeat, containing Aspergillus nidulans PacC site consensus sequences. This decameric repeat was able to mediate repression due to carbon and/or nitrogen sources as well as pH-dependent activation. A study in the context of trans-regulatory mutations in the Y. lipolytica RIM101 gene showed that the PacC-like sites, potential binding sites for YlRim101p, were implicated in the derepression of UAS2-driven expression at neutral-alkaline pH. The in vivo response of the PacC- like decamers to external pH was dependent on the status of the pH- regulated activator YlRim101p, which is homologous to the A. nidulans PacC regulator. The carbon/nitrogen regulation imposed on the decamers was shown to be independent of YlRim101p and to override its effects.

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