Genetic and biochemical studies of N-alkane non-ultilzing mutants of Saccharomycopsis lipolytica

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Abstract
Alkane non-utilizing mutants of the yeast Saccharomycopsis lipolytica were induced by ultraviolet light. Thirtyfour of the mutants were found to be alkaline-negative and fatty acid-positive (Phenotypes A and C) indicating a defect in n-alkane uptake or in hydroxylase complex activity. The hydroxylase complex is a microsomal aggregate composed of the first three enzymes of n-alkane catabolism. Leaky and non-mating mutants were eliminated leaving 21 mutants which were analyzed genetically. All 21 of the mutations showed a 1:1 pattern of segregation indicating they are chromosomal and all but one were recessive. Analyses of inter-mutant complementation and recombination showed that the 21 mutations represent 18 genes. Several of the mutants had pleiotropic phenotypes in addition to alkane non-utilization. These phenotypes included a loss of mating function, an inability to sporulate, a changed colony and cellular morphology, osmotic sensitivity and a lack of extracellular protease. The hydroxylase complex activities of mutants and wild type were assayed in cell-free extracts prepared by protoplast lysis. A small amount of detergent was necessary for the extraction of hydroxylase complex activity. The hydroxylase complex was inducible by n-decane and incubation was complete by 6 h. Hydroxylase complex activities in the mutants varied from 2.8% to 46.5% of wild type. The hydroxylase complex activities of two temperature sensitive mutants were as stable as wild type at the non-permissive temperature. These mutants showed temperature sensitive induction suggesting that the uptake of n-alkanes is temperature dependent in these strains.