A modified method of gene disruption in Komagataella phaffii with Cre/loxP system.

ABSTRACT

To increase protein production, technologies of gene manipulation for engineering the yeast Komagataella phaffii are extensively exploited. In this study, we developed a convenient gene disruption method in the yeast via Cre/loxP system. First, the simple gene disruption cassette [upstream homologous region (UP)-lox71-Sh ble-lox66-downstream homologous region (DW)] was constructed and transformed into the yeast to replace target gene. Second, the Sh ble gene of the cassette integrated in the chromosome was inserted with the auxiliary plasmid pPICZαA/cre/his4, resulting in an expanded cassette of UP-lox71-Sh ble-pPICZαA/cre/his4-lox66-DW. The auxiliary plasmid was generated via sequential insertion of cre and his4 genes into pPICZαA, and linearized with SmaI before its transformation. Finally, for deletion of the sequence between lox71 and lox66 sites in the expanded cassette, CRE protein responsible for Cre/loxP-mediated recombination was produced by methanol induction. Consequently, the corresponding sequence was eliminated permanently, only leaving a scar of lox72 site in the disrupted genes. This strategy was verified by disrupting two genes in the yeast. As the markers were recycled, it was also suitable for multiple gene disruption.