Fig. 2.

Comparison of conventional and Golden Gate based strain engineering strategies for P. pastoris. Overexpression of multiple genes (GOIs) in P. pastoris using conventional cloning plasmids requires several rounds of competent making (2 days), transformation (4 days including second streak-out) and clone screening (5 days) and takes at least 31 days for three genes with three selection markers. Alternatively, multiple vectors can be co-transformed at once, but resulting transformation efficiencies are usually very low and clonal variation increases. Appropriate flanking sites for the selection marker (loxP or FRT sites) enable marker recycling by recombinases (Cre or Flp, respectively), but require one more round of competent making and transformation which takes at least eight additional days. Golden Gate plasmids carry several transcription units at once and thereby enable transformation and screening in only nine days