Chloroplast engineering of Chlamydomonas reinhardtii to use phosphite as phosphorus source

Phosphorus (P) is a key biological element and a limiting nutrient in aquatic and terrestrial environments. The vast majority of organisms can only uptake phosphorus in its most oxidized form, as phosphate (PO₄ ³⁻), whereas a few prokaryotic species can metabolize phosphorus in a more reduced state such as phosphite (PO₃ ³⁻). Recently, it has been shown that by expressing the ptxD gene, encoding a NAD-dependent phosphite dehydrogenase (also known as phosphonate dehydrogenase), in Chlamydomonas reinhardtii phosphite utilization can be enabled. However, this was done by transforming the nuclear genome, where gene silencing is frequent and random integration of transgenes can result in variable levels of gene expression and pleiotropic effects. The aim of this work was to investigate if phosphite assimilation in the eukaryotic algae Chlamydomonas reinhardtii can also be achieved by expressing a codon-optimized ptxD gene in the chloroplast. To do this, the ptxD gene was targeted and stably integrated into the psbA exon 5-5S rRNA intergenic region within the inverted repeats of the C. reinhardtii chloroplast genome. Integration was shown to occur in the targeted site and transplastomic lines were shown to be homoplasmic and to stably accumulate the NAD-dependent phosphite dehydrogenase PTXD, enabling the cells to use phosphite as the sole phosphorus source in a 0.1–5 mM concentration range. This work demonstrates that transplastomic lines of C. reinhardtii expressing the prokaryotic ptxD gene can effectively be cultivated in phosphite, opening new opportunities for microalgae cultivation.