Highly efficient molecular delivery into microalgae by electroporation for enhancing biodiesel production

Abstract

Electroporation is a highly efficient molecules delivery method used for transformation in various cell types. However, in microalgae, there has been a lack of optimized electroporation parameters due to the presence of the cell wall, difficulties in protoplast preparation and maintaining cell viability. Therefore, this study was carried out to optimize the electroporation condition for transforming microalgae. Chlamydomonas reinhardtii strains of wild type (CC-124) and mutant (cell wall deficient) were used to investigate the effects of differently sized molecules, calcein (623 Da) and fluorescein isothiocyanate-dextran (FITC-dextran, 40 kDa), and applied to a wide range of electroporation parameters, voltage, and pulse length on cell viability and molecules uptake. In terms of viability, there was no significant difference in both strains on the applied electroporation parameters. In the case of small-sized molecules delivery, the effect of the cell wall was insignificant, as 1.25 kV/cm and 30 ms showed the highest efficiency for both strains, while the bigger-sized molecules were delivered well at 1.5 and 2.0 kV/cm and 30 ms for mutant and wild types, respectively. The optimized parameters of 1.5-2.0 kV/cm and 20-30 ms were used to deliver plasmid DNA (PtCrCFP) into wild-type, and it was observed that 1.25 kV/cm and 15 ms resulted in maximum transformants. Furthermore, it was found that cell viability had a more critical role than molecular delivery in gene transformation from the fact that a lower electric field revealed the highest electroporation efficiency. The integration and expression of PtCrCFP gene were examined, which revealed the existence of some hindrance to stable gene incorporation and fluorescent emission. The percentage of PtCrCFP gene integration and expression was 54.5 % (±4.42) and 8.6 %, respectively. The size of the plasmid (6 kbp) seemed to cause the low integration efficiency, and it was assumed that a strong chlorophyll background o...