Sequential deletion of amino acid permease genes in Saccharomyces cerevisiae to improve functional complementation assays

Saccharomyces cerevisiae mutants have been used since the early 1980s as a tool to clone genes from other organisms by functional complementation. This approach has been extremely successful in cloning for instance the first amino acid, sugar, urea, ammonium, peptide, potassium and ABC transporters in plants. Over the years, new strains with more mutations have been developed, enabling characterization of transport properties in a more precise way. In addition to a tool, these strains can be used to study the relative contribution of redundant transporters to the whole function. We focused on amino acid transport, starting with the yeast 22D8AA, developed to clone plant amino acid transporters in the early 2000s. We recently deleted two additional amino acid permeases, Gnp1 and Agp1, creating 22D10alpha. Sequencing of the 22D10alpha genome using long-read PacBio sequencing identified all expected mutations, and precisely characterized the nature of the deletions. Interestingly, 22D10alpha genotype was inaccurate, being Mata instead of Matalpha.