2Department of Biophysics, Ivan Franko Lviv National University, Lviv, Ukraine
3Institute of Chemistry and Chemical Technologies, Lviv National Polytechnic University, Lviv, Ukraine
4Biology Department, Concordia University, Montreal, Quebec, Canada
The genetic transformation of target cells is a key tool in modern biological research, as well as in many gene therapy and biotechnology applications. Here we describe a new method for delivery of DNA into several industrially important species of yeast, including Saccharomyces cerevisiae. Our method is based on the use of a novel nanoscale oligoelectrolyte polymer possessing a comb-like structure as a carrier molecule. Direct comparisons to standard transformation methods clearly show that our approach: (i) yields two times more transformants of Hansenula polymorpha NCYC 495 compared to electroporation approaches and 15 times more transformants compared to lithium acetate protocols, as well as (ii) 5 times more Pichia pastoris GS115 transformants compared to electroporation and 79 times more transformants compared to lithium acetate. Taken together, these results clearly indicate genetic transformation of yeasts using oligoelectrolyte polymer carriers is a highly effective means of gene delivery.
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).
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.
This comb-like polyampholitic polyelectrolyte was synthesized via controlled radical polymerization initiated by the oligoperoxide metal complex (OMC) in a polar organic media. OMC was coordinating Cu2+ complex of the copolymer composed of vinyl acetate, 5-tertbutylperoxy-5-methyl-1-hexene-3-yne, and maleic anhydride. Both the initial oligoperoxide and OMC derivate have been synthesized, as previously described (37-39).
Synthesis of the comb-like polyelectrolyte was carried out as follows: a monomer mixture of DMAEM (28.18 g (161.11 mol/l)) and VEP (3.19 g (15.31 mol/l)) was injected into round bottom glass reactor equipped with impeller mixer and backflow condenser. A solution of OMC (1.66 g (0.78 mol/l) in ethanol or dimethyl formamide (67 g (822.8 mol/l)) was then added with stirring. The incubation temperature was increased to 333 K, and the reactor was actively maintained at this temperature for 8 hrs under argon flow. The monomer conversion was controlled using a gravimetric technique; after achieving the desired degree of conversion (i.e., 60%), the solvent was evaporated under vacuum. Synthesized comb-like polyelectrolyte was purified by multiple precipitations from acetone solution into hexane, and dried under vacuum.
Synthesized polymer was dissolved in sterile distilled H2O, and the pH was adjusted to pH 7.4 (if not mentioned otherwise). A 1% water polymer solution was aliquoted into 1.5 mL plastic tubes (leaving as less air in the tube as possible), and stored at 4°C. Yeast strains, growth conditions, and media
Hansenula polymorpha NCYC 495 leu1–1, Pichia pastoris GS115 his4 and Saccharomyces cerevisiae BY4742 MATα his3Δ leu2Δ lysΔ ura3Δ strains were grown in YPD medium (1% yeast extract (Serva, Germany), 2% bacto-peptone (Serva, Germany), 2% glucose (Serva, Germany)) at 37°C (H. polymorpha) or 30°C (P. pastoris, S. cerevisiae), correspondingly.
Minimal modified Burkholder medium was used for selection of P. pastoris and S. cerevisiae transformants (40). For the selection of H. polymorpha transformants via geneticin (G418) resistance, 50 mg/L of G418 (Invitrogen, Sweden) was added to YPD plates. For the auxotrophic strains, amino acids L-leucine, L-uracil, L-histidine, L-lysine (Sigma-Aldrich, USA) were added to a final concentration of 40 mg/L.
DNA manipulation and transformation of Escherichia coli were carried out according to standard procedures (41). E. coli strains were grown in Luria-Bertani medium (LB) at 37°C supplemented with ampicillin (100 µg/mL, Sigma-Aldrich, USA), if necessary. EIectroporation assay
Electroporation was carried out by using a Bio-Rad Gene Pulser II, USA equipped with a Bio-Rad Pulse Controller II, USA. For yeast electroporation, a modified protocol from Becker and Guarente was used (4).
Transformants were counted 3–5 days after electroporation and statistical analysis (Student's t-test) was performed. Lithium acetate transformation
A modified protocol for delivering DNA into yeast cells following treatment with lithium acetate (LiAc) was used (30). After 3–5 days of growth, yeast transformants were counted and statistical analysis (Student's t-test) was performed. Yeast transformation using the developed polymer
H. polymorpha, P. pastoris and S. cerevisiae yeasts were grown overnight in non-selective YPD medium at 37°C (H. polymorpha) or 30°C (P. pastoris, S. cerevisiae). 150 µL of the overnight culture was transferred into 30 mL of YPD medium and grown until an OD600 ≤0.4–0.5. The cells were collected by centrifugation for 10 min at 3000 xg and then re-suspended in 100 µL of YPD. One µL of 1% water solution (pH 7.4) of oligoelectrolyte-based carrier BG-2 and 1 µg of plasmid DNA were added to the cell suspension, mixed gently and kept on ice for 45 min. Subsequently, cells were heat-shocked for 60–90 s at 42°C (H. polymorpha, S. cerevisiae) or 55°C (P. pastoris), chilled on ice for 2 min, and mixed with 1 mL of YPD medium. After incubation at 37°C (H. polymorpha) or 30°C (P. pastoris, S. cerevisiae) for 1 h, 100 µL of cells were plated on a selective medium and incubated at 37°C or 30°C, respectively. Yeast transformants were counted after 3–5 days of growth, and statistical analysis (Student's t-test) was applied. Stable transformation of Hansenula polymorpha
Yeast transformants were obtained using the protocol employed for transient transformation. After recovering and counting of the transformants, cells were re-plated several times on either selective medium or non-selective medium and grown as described for transient transformation. Stably transformed clones were counted. Statistical analysis
All experiments were repeated at least 3 times and in triplicate parallels. Results are presented as mean ± standard deviation. Statistical significance was evaluated by Student's t-test. Values of P<0.05 were considered statistically significant. Results and discussion
We synthesized our DNA carriers by grafting side cationic (dimethyl aminoethyl methacrylate and 5-(tertbutylperoxy)-5-methyl-1-hexen-3-yne)containing branches to an anionic carboxyl-containing backbone chain (Figure 1;Table 1). The carriers were designed to contain positively charged polymer branches to enable binding of DNA via negative charges on nucleotide phosphates. A dynamic light scattering study (Supplementary Figure S1) showed that after encapsulation of plasmid DNA, particle size increased from 19.2 to 95.6 nm. Formation of a particle-DNA complex was also confirmed by measuring zeta potential, which increased from 30.6 (particle alone) to 35.4 mV (particle and DNA complex).