Mar 31, 2020

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

BioRxiv : the Preprint Server for Biology
H. EdwardsPeng Xu

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 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...Continue Reading

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Mentioned in this Paper

Study
Immune Response
Immune System
Tuberculosis
Genes
Regulation of Biological Process
Pathogenic Organism
Transcription, Genetic
4-(methylthio)benzoic acid
Nucleic Acid Amplification Tests

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