Improved method for assembly of linear yeast expression cassettes using NEBuilder HiFi DNA Assembly Master Mix

Heterologous protein production in yeast expression systems (i.e., Kluyveromyces lactis and Pichia pastoris) normally involves insertion of a linear expression cassette into a target locus in the host genome (1-3). Typically, an expression cassette is assembled in E. coli by first cloning a gene of interest into a circular expression vector (Figure 1A). The expression cassette comprises DNA encoding a strong yeast promoter upstream of a heterologous gene of interest, a downstream transcription terminator sequence and a selectable marker gene. The entire cassette is flanked by locus-specific targeting sequences on either end (Figure 1B). The assembled vector is then amplified by propagation of the host E. coli cells, isolated by standard DNA preparation techniques, and subjected to restriction digestion to create a linear expression cassette. The linear fragment is then introduced into yeast whereby it integrates into a target locus on the host chromosome (Figure 1C).