Bacterial Lipoxygenases: versatile biocatalysts for fatty acid oxyfunctionalization

The transition to a biobased economy aims to reduce reliance on fossil fuels by converting biological materials, such as plant- and algae-derived fatty acids, into valuable compounds. Oxyfunctionalization, the process of introducing oxygen-containing groups, enhances fatty acid reactivity and enables the synthesis of diverse derivative products, with the modification site significantly influencing product properties.---Lipoxygenases (LOXs) are widespread oxyfunctionalization enzymes that are particularly promising due to their broad substrate range, distinct regioselectivities, and ability to function without costly coenzymes. Remarkably, bacterial LOXs have received little attention so far. Therefore, at the start of this thesis project, their occurrence and mode of action were systematically reviewed. Furthermore, bioinformatic studies revealed that bacterial LOXs can be categorized into nine distinct phylogenetic clusters, each characterized by specific structural and functional features.---To facilitate the biocatalytic exploitation of bacterial LOXs, the ferrous oxidation–xylenol orange (FOX) assay was optimized to enhance its suitability for screening LOX activity across a wide pH range with different polyunsaturated fatty acid (PUFA) substrates. The modified method proved suitable for high-throughput screening of LOX activity and effectively assessed substrate specificity.---Biochemical characterization of LOX from Microcystis aeruginosa (Ma LOX) revealed a unique dioxygenase pattern and dual dioxygenation activity, presumably facilitated by the shallow shape of its substrate-binding pocket. Beyond hydroperoxides, Ma-LOX produced epoxy alcohols and ketones, indicative for its HPI activity. This versatility underscores the biocatalytic potential of Ma-LOX.---Collectively, these findings establish bacterial LOXs as versatile biocatalysts for the production of flavors and fragrances, oleochemicals, and oxylipins. This work not only deepens our understanding of LOX enzymology but also paves the way for future advancements in harnessing bacterial LOXs for a wide range of biotechnological applications.---Subsequently, two bacterial LOXs were extensively characterized. In the case of Burkholderia thailandensis LOX (Bt-LOX), site-directed mutagenesis targeting residues at the bottom of the substrate-binding pocket and within the oxygen access channel allowed the identification of key residues governing its substrate preference and regioselectivity. Notably, one Bt-LOX variant enabled dioxygenation at the ω−2 position of ω-3 PUFAs, unveiling a novel LOX specificity. In addition to its dioxygenation activity, the hydroperoxide isomerase (HPI) activity of Bt-LOX was explored by modifying the enzymatic reaction conditions. Significant HPI activity was observed at high enzyme concentrations and under oxygen-limited conditions, providing precise control over Bt-LOX activity and offering insights into potential applications.