Isolation, cloning and expression analysis of two genes encoding pyruvate, orthophosphate dikinase (PPDK) in the green microalgae chlamydomonas reinhardtii

Recently, microalgae have attracted significant interest around the world due to their wide range of different applications in renewable energy, pharmaceutical, food and cosmetic industries. The green algae Chlamydomonas reinhardtii is a useful model of a photosynthetic cell and has been intensively used for studies of a number of physiological processes. Recently, several molecular technologies including nuclear and organellar transformation (nuclear, chloroplast and mitochondrial) systems, RNA interference, CRISPR etc. have been applied to C. reinhardtii. These attributes make C. reinhardtii an ideal algal organism to perform genetic and metabolic engineering of C. reinhardtii to increase oil accumulation in microalgae. At the moment a little information is available for molecular mechanisms that control oil accumulation in microalgae. C4 photosynthesis metabolic pathways (CO2 fixation by C4 metabolic pathway) are essential for high rates of carbon dioxide fixation in plants. High rates of photosynthesis are important for high biomass accumulation of algae. However, surprisingly C4 pathway enzymes and its regulatory factors have not been studied at the molecular level in any green algae, except the efforts which were focused on the molecular characterization of phosphoenolpyruvate carboxylase genes in C. reinhardtii. In this study, two Pyruvate, orthophosphate dikinase (PPDK) genes from green microalga C. reinhardtii were isolated and cloned for the first time and performed expression analysis at different growth conditions. We demonstrate that two PPDK genes encode functional active PPDK enzymes in C. reinhardtii and both genes are C- and N-responsive genes and up-/down-regulated by changes in [CO2] or [NH4+] during growth. Phylogenetic analysis of PPDK1 and PPDK2 and number of other PPDKs revealed a clustering into three general groupings: (i) the typical vascular-plant enzymes, of which CrPPDK2, CrPPDK1 and VcPPDK (Volvox carteri, closely related C. reinhardtii) are distant members; ii) bacterial and iii) protozoal PPDKs. CrPPDK2 shows more similarity with plant PPDKs, while CrPPDK1 has more similarity with VcPPDK. Notably, plant and algal PPDK proteins are more closely related to some protozoa than to bacteria. Collective findings provide key molecular insight into the PPDK1 and PPDK2 genes and corresponding PPDK enzymes in the eukaryotic algae. These studies will have a significant impact on applications for increasing algae biomass and altering oil storage levels versus protein storage in algae cells to produce oil-rich algae.