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

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 marker. Divalent lead supplemented in media induces lead sulfide (PbS) aggregation in MET25-deleted cells such that deficient cells grow brown/black, and cells with functional copies of MET25 grow white. It is hypothesized that the sulfide buildup from MET25 deficiency can also be exploited to neutralize and detoxify methylmercury by formation of mercuric sulfide (HgS) and ethanol, which confers toxic resistance to methyl-mercury. A plasmid-based expression system of the CRISPR-Cas12 nuclease coupled with crRNA targeting MET25, was also employed as a gene disruption technique to orthogonally demonstrate MET25 as a locus responsible for this phenotypic change and to test this locus as a target for our gene disruption platform. Kinetic growth curves of wild type and MET25-deficient cells were obtained under varying concentrations of methylmercury. Cellular toxicity to methyl mercury was calculated from the Hill equation. Furthermore, MET25 did not induce strict auxotrophic requirements for methionine in Y. lipolytica. Plasmid and chromosomal-based complementation of MET25 deficient cells on a double layer agar plate with gradients of lead and methionine demonstrates delayed phenotype restoration, indicating post-transcriptional regulation of methionine in this yeast.