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Marine algae produce complex polysaccharides, which can be degraded by marine heterotrophic bacteria utilizing carbohydrate-active enzymes. The red algal polysaccharide porphyran contains the methoxy sugar 6-O-methyl-D-galactose (G6Me). In the degradation of porphyran, oxidative demethylation of this monosaccharide towards D-galactose and formaldehyde occurs, which is catalyzed by a cytochrome P450 monooxygenase and its redox partners. In direct proximity to the genes encoding for the key enzymes of this oxidative demethylation, genes encoding for zinc-dependent alcohol dehydrogenases (ADHs) were identified, which seem to be conserved in porphyran utilizing marine Flavobacteriia. Considering the fact that dehydrogenases could play an auxiliary role in carbohydrate degradation, we aimed to elucidate the physiological role of these marine ADHs. Although our results reveal that the ADHs are not involved in formaldehyde detoxification, a knockout of the ADH gene causes a dramatic growth defect of Zobellia galactanivorans with G6Me as a substrate. This indicates that the ADH is required for G6Me utilization. Complete biochemical characterizations of the ADHs from Formosa agariphila KMM 3901T (FoADH) and Z. galactanivorans DsijT (ZoADH) were performed, and the substrate screening revealed that these enzymes preferentially convert aromatic aldehydes. Additionally, we elucidated the crystal structures of FoADH and ZoADH in complex with NAD+ and showed that the strict substrate specificity of these new auxiliary enzymes is based on a narrow active site.
Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.
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).