Isolation of oversecreting mutant strains of the yeast Yarrowia lypolytica

A random mutagenesis approach was utilized to isolate supersecreting mutants from the yeast strain, Yarrowia lipolytica YLASIn, using rice α-amylase as a marker for heterologous protein expression. Following mutagenic treatment with either ethylmethanesulfonic acid (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), six oversecreting mutants, designated YLOS (Y. lipolytica oversecretor), were recovered at a frequency of 1.2×10^-3 on the basis of their capacity to increase amylase yields in the culture supernatant by at least 2 fold relative to the parent strain. Isolation of these mutants was greatly facilitated using a rapid and simple screening method which allows the detection of amylase activity by the formation of transparent zones around colonies on starch-containing solid medium. By curing mutant cells of the plasmid and retransforming with a new copy of the rice α-amylase expression vector, the origin of the mutations in two supersecretors was determined to be chromosomal, based on their ability to retain the oversecreting phenotype. When Trichoderma reesei endoglucanase I was utilized as a marker for secretion, the levels of secreted enzyme were also enhanced, indicating that the effects of these supersecreting mutations are not specific for amylase. Northern blot analysis revealed that whereas YLOS 9 has a mutation on transcriptional level, YLOS 10 and 11 contain their mutations on a post-transcriptional level.