Isolation, characterisation and heterolog expression in Escherichia coli of A and B splicing variants of phosphoenolpyruvat carboxykinase 1 gene from Chlamydomonas reinhardtii

In this study, for the first time cloning, identification, characterization and expression analysis of Chlamydomonas reinhardtii Pck1 enzyme gene thought to be related to the level of oil content in algea, was aimed. Findings can be used to increase the oil content of algae in subsequent studies and to provide industrially high yield oil from algae. In the analyzes made, Chlamydomonas reinhardtii 2137 (CC-4886) strains were used as the source. Microalgae were grown in TAP medium under constant temperature and light conditions. Molecular analyzes were performed with nucleic acids and proteins obtained by extracting microalgae mass with liquid nitrogen. Pck1a and Pck1b, two different variants of the Pck1 gene were obtained by PCR amplification and cloned into E. coli and their activities were determined by enzyme activation measurements. Besides, sizes of enzyme proteins were shown by SDS-PAGE and Western Blot methods. Microalgae cells were grown at different nitrogen and carbon dioxide ratios on TAP medium and decreased enzyme activity in decreasing nitrogen ratios indicated that the ratio of algal oil could be increased if PCK1 enzyme was suspended. It has also been shown that increasing the carbon dioxide content increases the enzyme activity and that the enzyme PCK1 may be a photosynthetic enzyme, contributing to the understanding of the photosynthesis system in the model organism Chlamydomonas reinhardtii. Furthermore, the fact that Pck1 is associated with the photosynthesis system and another sign that silencing will cause an increase in the algal oil ratio. The demonstration of the enzyme PCK1 as an active enzyme in microalgae and the identification of the relation with the ratio of cellular oil in algae has made a significant contribution to the studies to increase the oil ratio in algae. Moreover, this characterization of the photosynthesis system in Chlamydomonas reinhardtii, a model organism, has been the starting point for the characterization of related enzyme genes in other closely related species, with reference to this study.