ABSTRACT

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.

摘要

将二氧化碳转化为有价值的化学品是对抗环境中二氧化碳浓度增加的有效策略。微藻通过光合作用产生有价值的化学物质，可用作生物燃料、工业材料、药用铅和食品添加剂的来源。因此，微藻技术的改进通过基因工程可能被证明有希望用于定制生产新的代谢物。对于微藻的转化，使用物理和机械技术（例如电穿孔、用 DNA 包被的微粒轰击和用玻璃珠涡旋）将核酸（例如质粒 DNA (pDNA)）递送到细胞中。然而，由于带负电荷的细胞壁和核酸之间的静电排斥，将核酸递送到微藻细胞中具有挑战性。为了解决这个问题，在本研究中，我们使用具有线性聚乙烯亚胺 (LPEI) 和 pDNA 的复合物通过电穿孔研究了微藻转化。然而，LPEI的高毒性降低了莱茵衣藻的转化效率细胞。我们发现 LPEI 的毒性是由于细胞摄取 LPEI 引起的氧化应激。为了抑制 LPEI 的毒性，抗氧化剂 2,2,6,6-四甲基哌啶-1-氧基（TEMPO）与 LPEI 共价结合；该偶联物被命名为 TEMPO-LPEI。有趣的是，TEMPO-LPEI 具有与 LPEI 相似的细胞摄取趋势，显着降低了氧化应激和细胞毒性。与使用裸 pDNA 和 LPEI/pDNA 复合物处理的那些相比，使用 TEMPO-LPEI 和 pDNA 复合物的电穿孔提高了转化效率。该结果表明与抗氧化剂结合的聚阳离子可用于促进微藻转化。