A genetic approach to unravel the starch degradation pathway and its regulation in Chlamydomonas

Miriam Schulz-Raffelt1, Vincent Chochois1, Laure Constans1, David Dauvillée2, Audrey Beyly1, Mélanie Solivérès2, Steven Ball2, Gilles Peltier1, and Laurent Cournac1

1)CEA Cadarache, DSV, Institut de Biologie Environnementale et de Biotechnologie, Laboratoire de Bioénergétique et Biotechnologie des Bactéries et Microalgues, UMR 6191 CNRS/CEA/Université d'Aix-Marseille, Saint-Paul-lez-Durance, F-13108 France 2) UMR 8576 CNRS/USTL, Université de Lille, 59655 Villeneuve d'Ascq, France

Sulfur deprivation is considered as the most efficient way to trigger long-term hydrogen photoproduction in unicellular green algae. Under deprivation conditions Chlamydomonas cells accumulate starch as carbohydrate storage and the resulting decrease in PSII allows anaerobiosis, which is essential for hydrogenase activity. Starch metabolism has been proposed as one of the major factors of hydrogen production, particularly during the PSII-independent (or indirect) pathway. While starch biosynthesis has been characterized in Chlamydomonas, little is known concerning starch degradation. In order to gain a better understanding of starch catabolism pathways and identify limiting steps of starch-dependent hydrogen production, we have designed a forward genetic screening procedure aimed at isolating mutants of Chlamydomonas affected in starch mobilization. Using to different screening protocols, the first one based on dark aerobic starch degradation and the second on light anaerobic starch degradation, eighteen mutants were isolated among a library of 15,000 insertion mutants, eight (std 1-8) with the first screen and ten (sda 1-10) with the second. Most of the strains showed a reduction or a delay in the PSII-independent hydrogen production. As further characterizing the mutant std1, the insertion was found to be localized in the DYRK2 gene. DYRK (dual-specificity tyrosine-phosphorylation regulated kinase) homologs have been found in mammals, invertebrates, plants and yeast. This protein kinase family appears to be involved in cellular growth and/or development. We report here on the first isolation of a DYRK homolog in the green lineage.

e-mail address of presenting author: miriam@schulz-raffelt.de