Insertional mutagenesis of AIDA or CYP720B1 in the green alga Chlamydomonas reinhardtii confers copper(II) tolerance and increased biomass

The widespread use of copper (Cu) in industrial and agricultural settings leads to the accumulation of excess Cu within aquatic ecosystems, posing a threat to organism health. Microalgal bioremediation has emerged as a popular and promising solution to mitigate the risks. Nevertheless, the genetic underpinnings and engineering tactics involved in heavy metal bioremediation by microalgae remain inadequately elucidated. In this study, two mutants obtained from screening a Chlamydomonas reinhardtii (C. reinhardtii) mutant library were identified as insertional mutagenesis in the AIDA (Cre12.g487450) and CYP720B1 (Cre10.g426700) genes. Interestingly, these two mutants exhibited decreased cell size and ciliary length but increased cell growth rates. Under Cu(II) stress, the AIDA and CYP720B1 mutants presented dose-dependent tolerance to Cu(II), resulting in increased biomass and improved cellular morphology. Furthermore, the analysis for the antioxidant system suggested that increased Cu(II) tolerance was associated with a low-level response strategy to Cu(II) stress. Transmission electron microscopy images also revealed increased stress-related organelles (starch granules, acidocalcisomes, and plastoglobules) in these two mutants. Considering the excellent Cu(II) tolerance and biomass of these two mutants, our findings provide potential microalgal strains for further genetic modifications and performance mining to improve aquatic Cu(II) bioremediation through biomass enhancement.