Characterization of Met25 as a Color Associated Genetic Marker inYarrowia lipolytica

Abstract

AbstractYarrowia lipolyticaoffers an ideal host for biosynthesis of high value natural products and oleochemicals through metabolic engineering despite being restricted to a limited number of selective markers, and counter-selection achieved primarily withURA3. In this work, we investigateMET25, a locus of sulfide housekeeping within the cell, to be exploited as a standard genetic marker. Divalent lead supplemented in media induces lead sulfide (PbS) aggregation inMET25-deficient cells such that deficient cells grow brown/black, and cells with functional copies ofMET25grow white. Loss ofMET25did not induce strict auxotrophic requirements for methionine inY.lipolytica, indicatingMET25deficiency could be rescued by alternative pathways. Plasmid and chromosomal-based complementation ofMET25deficient cells on a double layer agar plate with nutrient gradients demonstrates delayed phenotype (white morphology) restoration, indicating post-transcriptional feedback regulation of methionine biosynthesis in this yeast.MET25deficientY. lipolyticacould be used as an efficient whole-cell lead sensor with detection limit as low as 10 ppm of lead in drinking water. We further tested whetherMET25deficiency can be exploited to confer resistance to methyl-mercury through chemical neutralization and detoxification. Kinetic growth curves of wild type andMET25-deficient cells were obtained under varying concentrations of methylmercury and cellular toxicity to methyl mercury was calculated from the Hill equation. Our results indicate that methylmecury may not be used as the counter-selectable marker due to insignificant changes of growth fitness. This work demonstrates the utility of usingMET25as a sensitive lead sensor and the challenges of usingMET25as a counter-selectable genetic marker, as well as the complex regulation of methionine biosynthesis inY. lipolyitca, which may shed lights for us to develop valuable biotechnological applications centering around the sulfur house-keeping metabolism of the nonconventional yeast.