Portal de Periódicos da CAPES

High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol

2004; Future Science Ltd; Volume: 36; Issue: 1 Linguagem: Inglês

10.2144/04361dd02

1940-9818

Shixuan Wu, Geoffrey J. Letchworth,

CRISPR and Genetic Engineering

Transformation efficiencies for Pichia pastoris are usually several orders of magnitude below those for other yeast. We report here that pretreatment of P. pastoris with 0.1 M lithium acetate (LiAc) and 10 mM dithiothreitol (DTT) before electroporation increased transformation efficiency approximately 150-fold. DTT alone enhanced the transformation efficiency up to 20-fold, but LiAc alone had little effect. Cultures grown to 1.15–2.6 A at 600 nm had higher transformation efficiencies than younger or older cultures. A cell concentration of 10 10 /mL gave the highest efficiencies. Digestion of pPIC9K within the AOX1 gene with SacI gave efficiencies approximately 30 times higher than digestion in other genes with other enzymes. Given the optimization of these factors, the highest transformation efficiency was obtained with instrument settings of 1.5 kV, 25 µF, and 186 Ω The transformation efficiency at optimal conditions reached 4 × 10 6 transformants/µg DNA with pPIC9K. A maximum of 2.6 × 10 5 transformants was produced when 1 µg of pPIC9K DNA was used.