Yeast species have been used in a wide range of applications in modern biology, including in the production of various biological agents in industrial biotechnology, as experimental models for signaling processes taking place in higher eukaryotic cells, and as powerful model systems to characterize the molecular events associated with human mitochondrial and neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's (1). Delivery of DNA into yeast cells is the most critical, and limiting, step when it comes to the development of new strains to advance yeast-based research. Various approaches have been developed to facilitate delivery of nucleic acids into yeast cells, including lithium acetate (LiAc)-based methodologies, electroporation, gene gun transformation, protoplast transformation, and others (2-16). However, all these approaches present limitations, such as low delivery efficiency, high toxicity, durable consumption time, complexity of application, high cost of reagents and/or equipment. In addition, the two most commonly applied methods for DNA delivery into yeast cells – namely electroporation and LiAc chemical transformation are not efficient for some yeast species of high biotechnological interest (3, 9, 10, 16, 17). Yarrowia lipolitica, Dekkera bruxellensis (Brettanomyces bruxellensis), Phaffia rhodozyma (Xanthophyllomyces dendrorhous), Hansenula polymorpha, and Candida lipolitica are yeast species that possess high potential for biotechnology applications such as bio-ethanol production, yet present significant time, cost and efficiency challenges in their transformation with heterologous DNA using standard methods.

Significant progress in yeast transformation has been achieved through the use of specific nanoscale particles for DNA delivery (10, 18-21). Gene gun technology can be used to transform yeast species refractory to DNA delivery; however, this method requires expensive equipment, and is also both time consuming and known to provide relatively low transformation efficiency for some yeast species (6, 22-24).

Method summary

We describe a new method for rapid and efficient delivery of DNA into several industrially important yeast species using a novel oligoelectrolyte nanoscale polymer possessing a comb-like structure as the carrier.

Efficient yeast transformation methods are challenging due to the presence of the yeast cell wall, which composed mostly of mannose-containing proteins and glycans (25). Typical protocols for DNA delivery into yeast cells include time consuming and/or expensive steps to enzymatically remove cell wall components with lyticase or zymolyase; and chemical pretreatment of yeast cells with polyethylene glycol, lithium chloride, or thiol compounds (all these procedures make the cell wall leaky to macromolecules), or with 2-mercaptoethanol, dimethyl sulfoxide (DMSO), or dithiothreitol (DTT) (17, 26-34).

Polyethylene glycol (PEG) is the only polymeric agent presently used for yeast transformation with the LiAc-based method and protoplast transformation. PEG-based transformation of yeast protoplasts or spheroplasts where the cell wall has been removed is a time consuming and complicated method with irreproducible and often unsatisfactory results (33-36). Thus, the lack of convenient, efficient, and nontoxic method for DNA delivery remains one of the biggest challenges in yeast basic research and biotechnology.

Here, we propose a novel method for DNA delivery into yeast cells based on using a new nanoscale comb-like oligoelectrolyte polymer. This oligoelectrolyte polymer combines an anionic backbone with dimethyl aminoethyl methacrylate (DMAEM)-based side branches to enable effective DNA delivery into a variety of yeast species.

Materials and methods

Synthesis and characterization of comb-like oligoelectrolytes

The novel DNA carrier is a copolymer of a comb-like structure that combines an anionic type oligoelectrolyte chain, copolymer of vinyl acetate (VA), 5-(tertbutylperoxy)-5-methyl-1-hexen-3-yne (VEP), and maleic anhydride (MA), as a backbone and 1 to 3 grafted side chains, predominantly 2, of the cationic type, copolymer of DMAEM and VEP (Figure 1). The combination of these chains in carrier molecules provides tightly controlled solubility in a wide pH range, optimal surface activity, and the ability to form and stabilize nanoscale inter-polyelectrolyte complexes with DNA and their derivative water-based systems.

Figure 1.  General structure of the oligoelectrolyte polymeric carrier BG-2 (Click to enlarge)