Enzymatic Preparation and Regeneration of Protoplasts from the Red Microalga Porphyridium purpureum

Gene editing techniques are well-developed for model microalgae like Chlorella and Chlamydomonas. However, for the high-value species Porphyridium purpureum, its thick cell wall poses a significant barrier to genetic manipulation. Overcoming this challenge requires a robust method for preparing highly viable protoplasts. To generate protoplasts of P. purpureum, a targeted enzymatic lysis was applied based on the structural composition of the cell wall in this study. The results of single-factor experiments showed that a high protoplast yield (up to 43.55%) could be achieved under the following conditions: a total cellulase and macerozyme concentration of 2%, a cellulase-to-macerozyme ratio of 3:7, and a treatment time of 8 h in 0.2 mol/L KCl. Subsequently, Response Surface Methodology further optimized the enzymatic lysis conditions, yielding the optimal combination: a fixed combined concentration of cellulase and macerozyme at 2.2% in 0.2 mol/L KCL solution, a ratio of approximately 1:3, and an 8 h treatment, achieving a protoplast yield of 49.44%. Transmission electron microscopy, field emission scanning electron microscopy and Evans Blue staining confirmed that the protoplasts had smooth surfaces, no extracellular polysaccharide residues, and intact membranes and organelles. Furthermore, the optimal regeneration medium was ASW medium supplemented with 0.2 mol/L KCl, which might create a stable microenvironment for protoplast regeneration by balancing baseline osmotic pressure and ionic environment. This study established an effective protocol for protoplast preparation and regeneration, thereby facilitating the development of gene-editing tools for P. purpureum.