Genetic Engineering Tools for Saccharomyces cerevisiae

Book chapter, 2014

The yeast Saccharomyces cerevisiae is a widely used cell factory. Genetic engineering requires efficient transformation techniques, and many protocols for transformation of S. cerevisiae have been developed. These include methods involving spheroplast generation, electroporation, and biolistics with DNA coated microprojectiles. For expression of heterologous genes or overexpression of homologous genes in S. cerevisiae, plasmid vectors are usually employed. Most are shuttle vectors that allow cloning and amplification in Escherichia coli. Yeast plasmid vectors can be divided into different types. Probably the most widely used type of selectable markers are ones that confer prototrophy to auxotrophic strains containing mutations in amino acid and/or nucleotide biosynthetic pathways, e.g., LEU2 and URA3. Several of the prototrophic markers offer this possibility, the most widely used being the URA3 marker, which can be selected against using 5-fluoroorotic acid (5FOA). Proper transcription termination is a requirement for mRNA stability and thus also for high expression levels. Protein secretion requires the presence of an N-terminal signal sequence. Gene targeting is particularly efficient in S. cerevisiae because DNA double-strand breaks are preferentially repaired by homologous recombination as opposed to nonhomologous end joining. Since the codon composition at the gene’s 5' end seems to be most important, it may be sufficient only to optimize the first 14 to 32 codons to achieve good expression.