Elucidating the role of lipoxygenases in degrading the cell wall of Nannochloropsis oceanica

Abstract

Microalgae play a vital ecological and biotechnological role, offering sustainable solutions across various industries. Nannochloropsis oceanica is an emerging model for metabolic engineering and industrial applications. However, its robust algaenan-containing cell wall highly is recalcitrant and indigestible, posing a significant challenge for the extraction of valuable metabolites. An enzyme (NoLOX) identified in the proteome of N. oceanica may play a role in algaenan degradation. This thesis investigates the role of NoLOX in cell wall remodeling of N. oceanica. A heterologous expression system was developed in Escherichia coli to produce and biochemically characterize NoLOX in vitro. CRISPR/Cas9 genome editing was employed in an attempt to create loss of function NoLOX lines in N. oceanica. Overexpression strains with and without tdTomato fluorescent protein were constructed in N. oceanica to assess the effects of elevated NoLOX levels on cell wall integrity, morphology and algaenan monomer composition. Confocal imaging, flow cytometry and liquid chromatography-mass spectrometry were used to analyze these effects. Cell wall integrity was further evaluated by examining RNA and pigment extraction efficiency. The results indicate that overexpression of NoLOX weakens the cell wall, suggesting its involvement in algaenan degradation. Understanding algaenan degradation pathway not only advances our understanding of algal biology but also contributes for engineering Nannochloropsis strains with improved extractability of lipids and other high-value compounds, enhancing their biotechnological potential.