Functional screening of marine polysaccharide utilization loci – PULmarin

Marine polysaccharides represent the most abundant and the most diverse marine biomass. These macromolécules are biosynthesized mainly by photosynthetic organisms (e.g. algae, micro-algae, cyanobacteria) and constitute a major main carbon source for heterotrophs organisms. Except glycoaminoglycans (e.g. heparin, chondroitin), terrestrial polysaccharides do not carry sulfate ester groups in contrast with marine polysaccharides. Thus the sulfation of polysaccharides other than glycosaminoglycans is considered as a necessary adaptation and a marker of marine origin. Marine polysaccharides are also very appreciated in industry for their physicochemical properties (e.g. agarose, carrageenans, alginates). Despite their ecological role, economical importance and their potential for applications, the extent of the structural diversity of marine polysaccharides is not known.
During the last two decades, genome sequencing of marine organisms and of DNA from marine environmental samples (metagenomics) have generated a deluge of gene sequences; however, in contrast to terrestrial (meta)genomic investigations, the proportion of unknown protein families is higher in marine samples because oceans have been less studied so far. The functional interpretation of marine genomic and metagenomics data requires the prior functional characterization of genes/proteins to understand fundamental mechanisms encountered in marine environment such as organisms’ interactions, food-web, adaptation of populations to environmental change, etc. Interestingly, it was recently found that enzymes involved in the degradation of two marine polysaccharides (porphyran and cladophoran), were actually present in the human gut microbiota, suggesting that enzymes targeting other marine polysaccharides may be also found in this digestive system.
The degradation of marine polysaccharides requires the concerted action of enzymes cocktails comprising complementary activities (GH: glycoside hydrolases, PL: polysaccharides lyases, sulfatases, etc). In the case of bacteria belonging to the phylum Bacteroidetes, genes involved in the polysaccharide degradation are clustered on the genome and co-regulated in “polysaccharides utilization loci” (PULs), each PUL targeting a specific glycan structure. Several PULs comprising GHs, PLs and sulfatases potentially targeting marine polysaccharides are predicted environmental Bacteroidetes and in Bacteroidetes from the human gut microbiome. However, the actual targets of these PULs are unknown in absence of experimental evidence for the function (specificity) of the encoded enzymes.
We propose to systematically search and identify PUL putatively targeting marine polysaccharides in hundreds of genomes from sequence databanks as was already done for the PULs from 70 Bacteroidetes genomes from the human gut microbiota (www.cazy.org/PULDB). We will then select from marine and human gut Bacteroidetes about 50 PULs putatively targeting marine polysaccharides and which will represent approx. 500 genes encoding GHs, PLs, sulfatases, as well as proteins with unknown functions. All the proteins will be expressed recombinantly and will be biochemically characterized. At the end of the project we expect to have attributed the function to several marine PULs found in both marine organisms and in human gut microbiota. The results will directly impact the interpretation of marine genomic data and our understanding of the extent of the digestive abilities of the human gut microbiota.