Doctoral Thesis
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This dissertation focuses on the characterization of novel enzymes and metabolic pathways that fulfill crucial functions during marine carbohydrate degradation by Bacteroidetes and thus contributes to an advanced understanding of the global carbon cycle. Depolymerization and utilization of marine polysaccharides by Bacteroidetes requires a tremendous repertoire of enzymes with a wide range of functions. For instance, during the breakdown of the marine red algal polysaccharide porphyran, an oxidative demethylation of the methoxy sugar 6-O-methyl-D-galactose (G6Me) by cytochrome P450 monooxygenases occurs. This reaction produces huge amounts of cytotoxic formaldehyde, marine bacteria capable of degrading porphyran must therefore possess suitable formaldehyde detoxification pathways. Consequently, Article I focus on the identification of possible formaldehyde detoxification pathways in marine
Flavobacteriia, which led to the discovery of the ribulose monophosphate pathway as specific pathway for the detoxification of formaldehyde in certain Bacteroidetes like Zobellia galactanivorans. Furthermore, it was demonstrated in Article II that alcohol dehydrogenases play an essential role in the microbial utilization of G6Me and therefore possess a function in porphyran degradation. Discovering novel enzymes, entire enzymatic cascades or biotechnologically important microorganisms that can metabolize these marine carbohydrates also contributes to the utilization of marine polysaccharides as feedstock for potential biotechnological applications. A prospective biorefinery process was proposed in Article III by the identification of Bacillus licheniformis as promising utilizer of marine carbohydrate-derived monosaccharides and the creation of a microbial cell factory capable of growing on ulvan, a marine carbohydrate obtainable from algal bloom-dominating green algae, enabling an industrial use of the renewable and abundant algal biomass in future.
Marine algae are essential for fixation of carbon dioxide, which they transform into complex polysaccharides. These carbohydrates are degraded e.g., by marine Bacteroidetes and the understanding of their decomposition mechanism can expand our knowledge how marine biomasses can be accessed. This understanding then gains insights into the marine carbon
cycle. This thesis summarizes the current knowledge of marine enzymatic polysaccharide degradation in review Article I and extents a previously discovered ulvan degradation pathway in Article II with the description of a novel dehydratase involved in the ulvan degradation pathway. This enlarged ulvan-degradation pathway can be used to generate fermentable sugars from the algal derived polysaccharide ulvan. A potential biorefinery process is proposed in Article III, where B. licheniformis was engineered to degrade ulvan, thus establishing the initial steps for a microbial cell factory development. In addition to ulvan, also plenty of other complex carbohydrate sources are present in the ocean. The enzymatic elucidation principles previously developed were thus adapted towards a new marine carbohydrate. In Article IV a xylan utilization pathway was elucidated, using enzymes present in Flavimarina Hel_I_48 as model bacterium. The Flavimarina genome contains two separated genome clusters which potentially targets xylose containing polymers reflecting the diversity and adaptions towards different marine xylan-like substrates. Besides, marine Bacteroidetes are adapted towards decomposition of methylated polysaccharide, e.g., porphyran, via demethylation catalyzed by cytochrome P450 monooxygenases. This reaction results in the formation of toxic formaldehyde and thus the marine Bacteroidetes require formaldehyde detoxification principles. The analysis of potential formaldehyde detoxification mechanisms revealed a marine RuMP pathway (Article V) and a novel auxiliary activity of an alcohol dehydrogenase of which the encoding gene is adjacent to the demethylase cluster (Article VI).