Overlap Extension Construction and Direct Cloning of Loop-Out Prone DNA Expression Cassettes in Pichia pastoris:...

Overlap extension PCR (OE-PCR) is a simple technique allowing for the construction of linear expression cassettes for direct integration into the Komagataella phaffii genome. This strategy enables the assembly and amplification of expression cassettes of interest that are genetically unstable or cause physiological problems in Escherichia coli and accelerates the path from gene to yeast transformation. As a typical example, here we describe the cloning of the Secale cereale (rye) gene Sec2, coding for the prolamin 75k γ-secalin. This gene proved to be unstable in circular plasmids in E. coli; in silico analysis suggested a loop-out-prone secondary structure of the DNA due to repetitive sequences. In three individual reactions, overlapping DNA fragments of the expression cassette and the gene of interest were amplified, followed by a two-step PCR protocol joining these three fragments to a linear expression cassette. After purification, the linear expression construct was directly used for the transformation of K. phaffii.