Genetics, Vol. 151, 1379-1391, April 1999, Copyright © 1999

Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris

Monique A. Johnsona, Hans R. Waterhama, Galyna P. Ksheminskab, Liubov R. Fayurab, Joan Lin Cereghinoa, Oleh V. Stasyka, Marten Veenhuisc, Aleksander R. Kulachkovskyb, Andrei A. Sibirnyb, and James M. Cregga
a Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000,
b Institute of Biochemistry, Ukrainian Academy of Sciences, 290005 Lviv, Ukraine
c Department of Microbiology, University of Groningen, 9751 NN Haren, The Netherlands

Corresponding author: James M. Cregg, Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, P.O. Box 91000, Portland, OR 97291-1000., cregg{at}bmb.ogi.edu (E-mail)

Communicating editor: A. G. HINNEBUSCH

We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.




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