Characterization of Met25 as a Color Associated Genetic Marker in Yarrowia lipolytica

Abstract

Yarrowia lipolytica offers 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 with URA3. In this work, we investigate MET25, 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 in MET25-deficient cells such that deficient cells grow brown/black, and cells with functional copies of MET25 grow white. Loss of MET25 did not induce strict auxotrophic requirements for methionine in Y. lipolytica, indicating MET25 deficiency could be rescued by alternative pathways. Plasmid and chromosomal-based complementation of MET25 deficient 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. MET25 deficient Y. lipolytica could 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 whether MET25 deficiency can be exploited to confer resistance to methyl-mercury through chemical neutralization and detoxification. Kinetic growth curves of wild type and MET25-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 using MET25 as a sensitive lead sensor and the challenges of using MET25 as a counter-selectable genetic marker, as well as the complex regulation of methionine biosynthesis in Y. lipolyitca, which may shed lights for us to develop valuable biotechnological applications centering around the sulfur house-keeping metabolism of the nonconventional yeast.