Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism

Abstract

Transformation (i.e., genetic modification of a cell by the incorporation of exogenous DNA) is indispensable for manipulating fungi. Here, we review the transformation methods for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris and Aspergillus species and discuss some common modifications to improve transformation efficiency. We also present a model of the mechanism underlying S. cerevisiae transformation, based on recent reports and the mechanism of transfection in mammalian systems. This model predicts that DNA attaches to the cell wall and enters the cell via endocytotic membrane invagination, although how DNA reaches the nucleus is unknown. Polyethylene glycol is indispensable for successful transformation of intact cells and the attachment of DNA and also possibly acts on the membrane to increase the transformation efficiency. Both lithium acetate and heat shock, which enhance the transformation efficiency of intact cells but not that of spheroplasts, probably help DNA to pass through the cell wall.

Keywords: Saccharomyces cerevisiae; cell wall; electroporation; endocytosis; fungi; lithium acetate; polyethylene glycol; spheroplast; transfection; transformation.

Figure 1

Relative transformation efficiency (white bar) and frequency (grey bar) of the low-transformability mutants (A) and high-transformability mutants (B). The values are relative to those of the WT (BY4742), set at 1.0. The averages and minimum or maximum of 3 independent experiments are shown.

Figure 2

Putative mechanism of S. cerevisiae transformation. DNA initially attaches to the cell wall. PEG is indispensable for this attachment and for successful transformation of intact cells. PEG also possibly acts on the membrane to increase the transformation frequency and efficiency as well as the permeability to YOYO-1. The attached DNA passes through the cell wall. LiAc and heat shock help DNA to pass through the cell wall. DNA then enters into the cell via endocytotic membrane invagination. Some DNA in the endosomes is delivered to the vacuoles and digested. However, the manner in which DNA escapes digestion, reaches the nucleus and enters it through the nuclear pore is still unclear.