Pathway localization by fluorophore or epitope tagging can be accomplished through a multi-staged DNA construct and confirmation process, to generate a series of successfully tagged protein targets. Prerequisite conditions for this process in Y. lipolytica are auxotrophic selection (leu2 or ura3), impaired non-homologous end joining by deletion or impairment of ku70, and plasmids or gene pieces for epitope-selection cassette construction. The general approach for gene tagging can work for C- or N-terminal tags. Gene overexpression from an episomal plasmid can be accomplished through transcript amplification and cloning. C-terminal tagging allows expression of a gene-GFP fusion to be regulated from the endogenous promoter. The epitope-selection cassette also includes a constitutive or highly expressed promoter driving the auxotrophic or other selectable marker gene such as one conferring antifungal or antibiotic resistance. Strains for pathway localization utilize overlap PCR, PEG-based transformation, and a fast DNA preparation for rapid colony screening. Successful transformants can be used for pathway localization and condition-specific response analysis.
Keywords: Endogenous; Gibson cloning; Overexpression; Overlap PCR; Pathway localization; Primer; Transformation.