Insertional mutagenesis in the n-alkane-assimilating yeast Yarrowia lipolytica: Generation of tagged mutations in genes involved in hydrophobic substrate utilization

Tagged mutants affected in the degradation of hydrophobic compounds (HC) we re generated by insertion of a zeta-URA3 mutagenesis cassette (MTC) into th e genome of a zeta-free and ura3 deletion-containing strain of Yarrowia lip olytica. MTC integration occurred predominantly at random by nonhomologous recombination. A total of 8,600 Ura(+) transformants were tested by replica plating for (i) growth on minimal media with alkanes of different chain le ngths (decane, dodecane, and hexadecane), oleic acid, tributyrin, or ethano l as the C source and (ii) colonial defects on different glucose-containing media (YPD, YNBD, and YNBcas). A total of 257 mutants were obtained, of wh ich about 70 were affected in HC degradation, representing different types of non-alkane-utilizing (Alk(-)) mutants (phenotypic classes alkA to alkE) and tributyrin degradation mutants. Among Alk- mutants, growth defects depe nding on the alkane chain length were observed (alkAa to alkAc). Furthermor e, mutants defective in yeast-hypha transition and ethanol utilization and selected auxotrophic mutants were isolated. Flanking borders of the integra ted MTC were sequenced to identify the disrupted genes. Sequence analysis i ndicated that the MTC was integrated in the LEU1 locus in N083, a leucine-a uxotrophic mutant, in the isocitrate dehydrogenase gene of N156 (alkE leaky ), in the thioredoxin reductase gene in N040 (alkAc), and in a peroxine gen e (PEX14) in N078 (alkD). This indicates that MTC integration is a powerful tool for generating and analyzing tagged mutants in Y. lipolytica.