Alpha-glucans from bacterial necromass indicate an intra-population loop within the marine carbon cycle.

Phytoplankton blooms provoke bacterioplankton blooms, from which bacterial biomass (necromass) is released via increased zooplankton grazing and viral lysis. While bacterial consumption of algal biomass during blooms is well-studied, little is known about the concurrent recycling of these substantial amounts of bacterial necromass. We demonstrate that bacterial biomass, such as bacterial alpha-glucan storage polysaccharides, generated from the consumption of algal organic matter, is reused and thus itself a major bacterial carbon source in vitro and during a diatom-dominated bloom. We highlight conserved enzymes and binding proteins of dominant bloom-responder clades that are presumably involved in the recycling of bacterial alpha-glucan by members of the bacterial community. We furthermore demonstrate that the corresponding protein machineries can be specifically induced by extracted alpha-glucan-rich bacterial polysaccharide extracts. This recycling of bacterial necromass likely constitutes a large-scale intra-population energy conservation mechanism that keeps substantial amounts of carbon in a dedicated part of the microbial loop.

Marine Bacteroidetes enzymatically digest xylans from terrestrial plants.

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 ß-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.

Unique alcohol dehydrogenases involved in algal sugar utilization by marine bacteria.

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. KEY POINTS: • Knockout of the ADH-encoding gene revealed its role in 6-O-methyl-D-galactose utilization, suggesting a new auxiliary activity in marine carbohydrate degradation. • Complete enzyme characterization indicated no function in a subsequent reaction of the oxidative demethylation, such as formaldehyde detoxification. • These marine ADHs preferentially convert aromatic compounds, and their strict substrate specificity is based on a narrow active site.

Metabolic engineering enables Bacillus licheniformis to grow on the marine polysaccharide ulvan.

BACKGROUND: Marine algae are responsible for half of the global primary production, converting carbon dioxide into organic compounds like carbohydrates. Particularly in eutrophic waters, they can grow into massive algal blooms. This polysaccharide rich biomass represents a cheap and abundant renewable carbon source. In nature, the diverse group of polysaccharides is decomposed by highly specialized microbial catabolic systems. We elucidated the complete degradation pathway of the green algae-specific polysaccharide ulvan in previous studies using a toolbox of enzymes discovered in the marine flavobacterium Formosa agariphila and recombinantly expressed in Escherichia coli. RESULTS: In this study we show that ulvan from algal biomass can be used as feedstock for a biotechnological production strain using recombinantly expressed carbohydrate-active enzymes. We demonstrate that Bacillus licheniformis is able to grow on ulvan-derived xylose-containing oligosaccharides. Comparative growth experiments with different ulvan hydrolysates and physiological proteogenomic analyses indicated that analogues of the F. agariphila ulvan lyase and an unsaturated ß-glucuronylhydrolase are missing in B. licheniformis. We reveal that the heterologous expression of these two marine enzymes in B. licheniformis enables an efficient conversion of the algal polysaccharide ulvan as carbon and energy source. CONCLUSION: Our data demonstrate the physiological capability of the industrially relevant bacterium B. licheniformis to grow on ulvan. We present a metabolic engineering strategy to enable ulvan-based biorefinery processes using this bacterial cell factory. With this study, we provide a stepping stone for the development of future bioprocesses with Bacillus using the abundant marine renewable carbon source ulvan.

Connecting Algal Polysaccharide Degradation to Formaldehyde Detoxification.

Formaldehyde is a toxic metabolite that is formed in large quantities during bacterial utilization of the methoxy sugar 6-O-methyl-d-galactose, an abundant monosaccharide in the red algal polysaccharide porphyran. Marine bacteria capable of metabolizing porphyran must therefore possess suitable detoxification systems for formaldehyde. We demonstrate here that detoxification of formaldehyde in the marine Flavobacterium Zobellia galactanivorans proceeds via the ribulose monophosphate pathway. Simultaneously, we show that the genes encoding the key enzymes of this pathway are important for maintaining high formaldehyde resistance. Additionally, these genes are upregulated in the presence of porphyran, allowing us to connect porphyran degradation to the detoxification of formed formaldehyde.

A new carbohydrate-active oligosaccharide dehydratase is involved in the degradation of ulvan.

Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Δ-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoA-Rha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharides into monosaccharides.

Marine Polysaccharides: Occurrence, Enzymatic Degradation and Utilization.

Macroalgae species are fast growing and their polysaccharides are already used as food ingredient due to their properties as hydrocolloids or they have potential high value bioactivity. The degradation of these valuable polysaccharides to access the sugar components has remained mostly unexplored so far. One reason is the high structural complexity of algal polysaccharides, but also the need for suitable enzyme cocktails to obtain oligo- and monosaccharides. Among them, there are several rare sugars with high value. Recently, considerable progress was made in the discovery of highly specific carbohydrate-active enzymes able to decompose complex marine carbohydrates such as carrageenan, laminarin, agar, porphyran and ulvan. This minireview summarizes these achievements and highlights potential applications of the now accessible abundant renewable resource of marine polysaccharides.