Reduced cytotoxicity of polyethyleneimine by covalent modification of antioxidant and its application to microalgal transformation

The conversion of carbon dioxide into valuable chemicals is an effective strategy for combating augmented concentrations of carbon dioxide in the environment. Microalgae photosynthetically produce valuable chemicals that are used as biofuels, sources for industrial materials, medicinal leads, and food additives. Thus, improvements in microalgal technology via genetic engineering may prove to be promising for the tailored production of novel metabolites. For the transformation of microalgae, nucleic acids such as plasmid DNA (pDNA) are delivered into the cells using physical and mechanical techniques, such as electroporation, bombardment with DNA-coated microprojectiles, and vortexing with glass beads. However, owing to the electrostatic repulsion between negatively charged cell walls and nucleic acids, the delivery of nucleic acids into the microalgal cells is challenging. To solve this issue, in this study, we investigated microalgal transformation via electroporation using polyplexes with linear polyethyleneimine (LPEI) and pDNA. However, the high toxicity of LPEI decreased the transformation efficiency in Chlamydomonas reinhardtii cells. We revealed that the toxicity of LPEI was due to oxidative stress resulting from the cellular uptake of LPEI. To suppress the toxicity of LPEI, an antioxidant, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), was covalently conjugated with LPEI; the conjugate was named as TEMPO-LPEI. Interestingly, with a cellular uptake tendency similar to that of LPEI, TEMPO-LPEI dramatically decreased oxidative stress and cytotoxicity. Electroporation using polyplexes of TEMPO-LPEI and pDNA enhanced the transformation efficiency, compared to those treated with bare pDNA and polyplexes of LPEI/pDNA. This result indicates that polycations conjugated with antioxidants could be useful in facilitating microalgal transformation