1 Metabolomic profiling reveals developmentally regulated biosynthesis of polyprenols and dolichols in the malaria parasite Flavia M. Zimbres 1,2# , Ana Lisa Valenciano 1,2# , Emilio F. Merino 1,2 , Nicole R. Holderman 1 , Anat Florentin 3,2 , Guijuan He 4 , Katarzyna Gawarecka 5 , Karolina Skorupinska-Tudek 5 , Maria L. Fernández-Murga 6 , Ewa Swiezewska 5 , Xiaofeng Wang 4 , Vasant Muralidharan 3,2 , Maria Belen Cassera 1,2* From the 1 Department of Biochemistry & Molecular Biology, University of Georgia, Athens GA 30602; 2 Center for Tropical and Emerging Global Diseases (CTEGD), University of Georgia, Athens GA 30602; 3 Department of Cellular Biology, University of Georgia, Athens GA 30602; 4 School of Plant and Environmental Sciences, Virginia Tech, Blacksburg VA 24061; 5 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland; 6 Laboratory of Experimental Pathology, Health Research Institute Hospital La Fe, Valencia 46026, Spain Running title: Biosynthesis of cis-polyisoprenols in P. falciparum # Contributed equally to this work * To whom correspondence should be addressed: Maria Belen Cassera: Department of Biochemistry & Molecular Biology and Center for Tropical and Emerging Global Diseases (CTEGD), University of Georgia, Athens GA 30602; maria.cassera@uga.edu; Tel. (706) 542-5192. Keywords: Plasmodium, malaria, polyprenol, dolichol, polyprenol reductase, SRD5A3, LC-HRMS ABSTRACT The cis-polyisoprenoid lipids namely polyprenols (POH), dolichols (DOH) and their derivatives are linear polymers of several isoprene units and have recently gained special attention due to several breakthroughs in the field including improvements in the analytical techniques for their analysis, partial identification of their enzymatic machinery, and the discovery of new biological functions beyond glycosylation. In eukaryotes, POH and DOH are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, POH are hardly detectable in eukaryotic cells under normal conditions with the exception of plants and sporulating yeast. Our metabolomics studies revealed that cis- polyisoprenoids are more prevalent and diverse in the protozoan parasite Plasmodium falciparum than previously postulated as we uncovered the active biosynthesis of medium-long POH and DOH (15 to 19 isoprene units). Moreover, a distinctive POH and DOH profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed. These results suggest that cis- polyisoprenoid biosynthesis is developmentally regulated. In addition, we confirmed that DOH biosynthesis occurs via reduction of the POH to DOH by an active polyprenol reductase (PfPPRD) and metabolomics analyses of a PfPPRD conditional mutant suggest that a salvage mechanism of DOH may exist. Malaria is caused by protozoan parasites of the genus Plasmodium and most cases of life- threatening malaria are attributable to infection with Plasmodium falciparum. The parasite has a complex life cycle that involves the human host and its vector, the Anopheles mosquitoes. All clinical features are caused during the asexual intraerythrocytic life cycle due to the repeated invasion of human red blood cells (RBCs). The intraerythrocytic cycle of P. falciparum lasts around 48 h, during which the parasite progresses through four morphologically different stages: ring, trophozoite, and schizont stages, ending with the rupture of the erythrocyte and release of merozoites that will invade new erythrocytes. Transmission of the malaria parasite requires the development of male and female gametocytes (gametocytogenesis), certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not this version posted July 11, 2019. . https://doi.org/10.1101/698993 doi: bioRxiv preprint