The DNA binding capability of these carriers (Supplementary Figure S2) was evaluated by an electrophoretic method that monitors shifts in plasmid DNA band positions. The results of this electrophoretic analysis suggest formation of ion bonds between the polymer carrier and plasmid DNA. Polymeric carrier BG-2 exhibited the optimal set of indicators of DNA binding and yeast transformation, and was therefore selected for further use in this study.
For yeast transformation experiments, we used Hansenula polymorpha and Pichia pastoris which are known for high biotechnological potential, however, their genetic transformation shows low efficiency. We compared transformation efficiencies among three different methods: (i) the chemical-based LiAc method, (ii) electroporation, and (iii) the novel method we describe here based on using the oligoelectrolyte carrier. Transformation of Hansenula polymorpha yeasts
Hansenula polymorpha is widely used in the production of bio-pharmaceuticals, genetically modified enzymes, vaccines, and other compounds of high industrial and medical importance (42-44). Although these yeasts exhibit many unique properties, their genetic engineering is limited due to low transformation efficiency when using the LiAc method (45). Therefore, electroporation is the most often used for genetic transformation of this yeast species, a technique that tends to be time consuming (necessitating competent cell preparation prior to electroporation), requires additional equipment, and frequently fails.
To test our new BG-2 polymer-based transformation method, we sought to transform the circular pGLG578 (contained LEU2 gene of Saccharomyces cerevisiae as a selectable marker) or linear HindIII-digested pYT3 (contained LEU2 gene of Saccharomyces cerevisiae as a selectable marker) plasmid (Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine) into H. polymorpha NCYC 495 leu1-1 yeast using the traditional LiAc method, electroporation, and the polymer-based technique. Leu+ and geneticineR transformants were selected on the YPD media supplemented with G418 (50 mg/L) antibiotic without leucine when pGLG578 plasmid was used. When pYT3 plasmid was used, Leu+ transformants were selected on solid minimal modified Burkholder media without leucine. The transformants were selected for leucine independence and G418 resistance.
Efficient transformation of this yeast strain using our method was usually achieved within 4 h, faster than either LiAc transformation (5 h) or electroporation (7 h). Additionally, our transformation protocol resulted in two times more transient transformants than electroporation, and 15.7 times more transformants than LiAc (Figure 2). It should be noted that both the electroporation and LiAc methods target only non-native cells that are made competent for transformation in a time consuming pre-treatment procedure. On the other hand, our method allows efficient transformation of native fast-growing log-phase yeast cells without any pre-treatments.
Application of our method also enabled more efficient delivery of non-linearized plasmid DNA as compared with the above mentioned classic methods (Figure 3). In addition, we tested the different methods for stable genetic transformation of H. polymorpha NCYC 495 yeast. The use of our method resulted in the recovery of 170 stably transformed clones, comparing to 120 clones recovered when electroporation was used and 20 clones recovered from the LiAc method (Figure 4). All genetically transformed stable yeast clones were selected for leucine independence or/and for G418 resistance. A useful property for ascertaining that characteristic is an expression of a new phenotype that is caused by the plasmid presence. The presence of DNA insert has been confirmed by the PCR (data are not shown). The primers used in this study are listed in the Supplementary Table S1.
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.