A Penicillium chrysogenum gene (aox) identified by specific induction upon shifting pH encodes for a protein which shows high homology to fungal alcohol oxidases

Abstract.

Parallel cultures of Penicillium chrysogenum were grown in controlled bioreactors under conditions of penicillin production and one was shifted from the initial pH 6.0 to pH 8.0. RNA was isolated from both cultures and used for a differential hybridization experiment to identify genes specifically induced upon this pH shift. About 2,000 plaques of a cDNA library constructed from pH 8.0 material were screened with a pH 8.0 to pH 6.0 subtracted probe. Two independent clones of which the RNA was highly abundant in pH 8-shifted material and essentially not present in pH 6 material were isolated. Both clones were found to belong to one specific gene, which could be identified by sequence homology as an alcohol oxidase. The identified aox gene encoded for a peptide of 666 amino acids, interrupted by nine introns; and it showed high homology (>65% identity) to alcohol oxidases from Cladosporium fulvum and the methanol-utilizing yeasts Candida boidinii, Hansenula polymorpha (now Pichia angusta) and P. pastoris. The transcription start was identified by primer extension analysis. Southern analysis revealed that related genes are widely distributed among fungal species.