Figure 3.  Transformants number ofHansenula polymorpha NCYC 495 leu1-1yeast when using different transformation methods with a circular plasmid. (Click to enlarge)

Figure 4.  Stable transformants number ofHansenula polymorpha NCYC 495 leu1-1yeast. (Click to enlarge)

Transformation of Pichia pastoris yeast

Pichia pastoris is well known for its use in the biotechnology sector. It is one of the most attractive microorganisms currently used for the expression of heterologous eukaryotic proteins under the constitutive and inducible promoters (46-51).

Preliminary experiments indicated that we were able to transform P. pastoris GS115 his4 yeast with a pPIC3.5 (7.8 kb) plasmid DNA (containing HIS4 gene as a selectable marker) (Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine) under conditions shown previously to be optimal for H. polymorpha. His⁺ transformants of P. pastoris were selected on a solid minimal modified Burkholder medium without histidine. Transformants were then selected for histidine independence.

Although transformation was possible using these conditions, transformation efficiency with P. pastoris was not sufficiently high in these experiments. However, we found that elevation of the heat shock temperature from 42 °C to 55 °C led to a significant increase in genetic transformation of H. polymorpha.

Thus, the method developed for genetic transformation of H. polymorpha and P. pastoris yeasts is more convenient, faster, and efficient than other currently used methods. This new transformation method resulted in five times more transient transformants than the electroporation, and 79 times more transformants than the LiAc method (Figure 5). Perhaps as important, our method of yeast transformation does not require the use of an electroporator or any other technical devices.

Figure 5.  Transformants number ofPichia pastoris GS115 his4yeast when using different transformation methods. (Click to enlarge)

Branched polyethylenimine (PEI) might be considered the closest commercially available analog to the polymeric carrier developed here. PEI has been widely used for DNA delivery in the mammalian cells (52-55). The mechanisms of action of the PEI, as well as our developed polymer, might be the following: (i) condensing DNA, (ii) providing protection against potential nuclease degradation, and (iii) promoting association with the plasma membrane of a target cell to enhance entry into the cell. Following an uptake, DNA should be capable of escape from the endosome and be released into the cytoplasm to be capable of reaching the nucleus. We found that PEI exhibited significantly lower transformation efficiencies with yeast cells, compared with our oligoelectrolyte polymeric nanoscale carriers.

Transformation of Saccharomyces cerevisiae yeast

Saccharomyces cerevisiae yeast is a unicellular eukaryotic organism most intensively used and studied in molecular and cell biology; in addition, it is widely used in biotechnology (6, 7, 16, 56). Although current transformation methodologies are enough sufficient for S. cerevisiae, we were interested to see if our oligoeletrolyte polymeric carriers could further enhance transformation efficiency.

Comparison of transformation efficiency using a circular episomal plasmid DNA (pYEp352 of 5.2 kb (containing URA3 gene as a selectable marker), Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine) and yeast Saccharomyces cerevisiae BY4742 MATα his3Δ leu2Δ lysΔ ura3Δ cells were performed. Ura⁺ transformants of S. cerevisiae was selected on a solid minimal modified Burkholder medium without uracil with transformants selected based on uracil independence.

Our polymeric carrier based transformation approach yielded 17,100 colonies of yeast transformants per 1 µg of DNA, while the standard electroporation protocol resulted in 1000 colonies, and chemical LiAc-based transformation yielded 8300 colonies (Figure 6).

Figure 6.  Transformants number ofSaccharomyces cerevisiaeBY4742 yeast when using different transformation methods. (Click to enlarge)

Thus, there is no significant increase in the transformation efficiency when using our carrier with S. cerevisiae cells whose surface is well permeable to plasmid DNA. However, a distinct advantage in the transformation efficiency could be demonstrated when using yeast species such as Hansenula polymorpha and Pichia pastoris which have proven less emmenable to transformation in previous studies. These findings strongly suggest that the developed polymeric carrier possesses chemical characteristics necessary for efficient DNA delivery. We can hypothesize that such characteristics could include the hydrophobic core chain required for the improved penetration through the plasma membrane or possibly the positively charged polymer branches involved in binding DNA molecule, although this remains to be further investigated.