Chlamydomonas reinhardtii purivat ortofosfat(pi) Dikinase(PPDK) 1 geninin Escherichia coli'de klonlanması, karakterizasyonu ve heterolog ekspresyonu

Given that fossil fuel resources are limited, the researchers look for alternatives. Algae is the most promising alternative and offers distinct advantages over traditional oil crops. However, studies on lipid metabolism in eukaryotic, single-celled, photosynthetic microalgae are limited. Chlamydomonas reinhardtii is a well-studied representative of eukaryotic microalgae that has been used as a model organism for a number of physiological, biochemical and genetic studies for more than a decade. However, surprisingly enzymes of C4-metabolism and regulatory factors have not been studied in C. reinhardtii at the molecular level, except the recent effort, which was focused on the molecular characterization of phosphoenolpyruvate carboxylase (PEPC) genes(Ppc 1 and Ppc2) in C. reinhardii (Mamedov et al., 2005; Moellering et al., 2007; Mamedov and Chollet, 2010). Thus, study of enzymes of C4-metabolism in the photosynthetic model organism C. reinhardtii would be very important for algal biomass accumulation, hence for biofuel. Pyruvate, phosphate dikinase (PPDK, EC 2.7.9.1) is important enzyme in the pathway of photosynthetic CO2 fixation in C4 plants and in some CAM plants, which catalyzes the ATP- and Pi-dependent formation of phosphoenolpyruvate (PEP), the primary CO2 acceptor molecule, from pyruvate. In this study, for the first time, we successfully isolated and cloned PPDK (CrPPDk) gene from cDNA of Chlamydomonas reinhardtii. Our results confirmed that CrPPDK gene transcribed in vivo and encodes a fully active, recombinant PEPC in E.coli. Gene expression analyses demonstrate that CrPPDK gene is CO2 or NH4 + responsive gene and its steady-state transcript levels are up-/down-regulated by varying levels of CO2 or NH4 + supplied to the growth medium.