Positive selection of novel peroxisome biogenesis-defective mutants of theyeast Pichia pastoris

We have developed two novel schemes for the direct selection of peroxisome- biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pasto ris Both schemes take advantage of our observation that methanol-induced pe x mutants contain little or no alcohol oxidase (AOX) activity. AOX is a per oxisomal matrix enzyme that catalyzes the first step in the methanol-utiliz ation pathway. One scheme utilizes allyl alcohol, a compound that is not to xic 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 s electively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defe ctive in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme requir ed to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld 1 strains are sensitive to methanol because of the accumulation of formalde hyde. However, fld1 per mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methan ol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously uniden tified PEX groups.