Fatty acid oxidizing enzymes in Lobosphaera incisa

by Benjamin Djian
Doctoral thesis
Date of Examination:2017-05-03
Date of issue:2018-04-23
Advisor:Prof. Dr. Ivo Feussner
Referee:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Franc Meyer
Referee:Prof. Dr. Ralf Ficner
Referee:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Jörg Stülke
Persistent Address: http://dx.doi.org/10.53846/goediss-6847

 

 

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Abstract

English

Green microalgae are a model in biotechnology because of their capacity to accumulate neutral lipids, as part of a general lipid remodeling mechanism under stress. The species L. incisa is of special interest for its unique TAG acyl chain composition, especially 20:4 (n-6) that can reach up to 21 % of dry weight after nitrogen starvation. In order to identify lead actors of this remodeling, special focus was put on lipid oxidizing processes, which have been described to be of major importance in flowering plants. Investigation of the available genome, transcriptome and proteome of L. incisa allowed the identification of a putative LOX, which so far has not been studied in green microalgae. The identification as a LOX was first confirmed in silico, by alignments and 3D structure prediction. The heterologous expression of LiLOX in E. coli was achieved in mg quantities, allowing further characterization of the LOX via enzymatic assays in vitro. Throughout the study, LiLOX was found to be a good model to study plastidic plant LOXs, as it shares highest identity with this class of enzymes and was found to have identical regio- and stereospecificity. This hypothesis was further confirmed by the localization of LiLOX in plastids, investigated via transient expression in epithelial onion cells. In order to further characterize this class of enzymes, mutational studies and attempts at crystallizations were performed and are presented in this thesis. An important part of this work focused on identification of the endogenous substrate of LiLOX. In this regards, an ex vivo enzymatic assay, coupled with non-targeted analysis via mass spectrometry allowed the identification of MGDG, DGDG and PC as three substrate candidates, later confirmed via in vitro assays. Further investigation revealed that LiLOX has preferences towards the lipid class MGDG, which seems in agreement with its localization in the galactolipid rich plastid. Altogether, this study shows the first characterization of plastidic LOX from green algae, showing preference for MGDGs. Finally, this study targeted the role of LiLOX in the general lipid remodeling taking place in L. incisa during nitrogen starvation. Since this remodeling is accompanied with the degradation of the plastidic membrane, the involvement of LiLOX in this process was discussed.
Keywords: Plastid; Lipoxygenase; Microalgae; MGDG; Membrane; Oxidation; Galactolipid
 
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