Investigating Microbial Utilisation of Seaweed Cell Wall Polysaccharides

<p dir="ltr">Seaweed cell wall polysaccharides are an invaluable reservoir for photosynthetically fixed carbon in the marine environment. This thesis interrogates the genomic and enzymological components that enable two recently isolated seaweed-associated bacteria, <i>Rhizobium</i> sp. nov. C1 (<i>Rhi</i>C1) and <i>Aliiglaciecola</i> sp. nov. SL4 (<i>Gla</i>SL4) to access this crucial nutrient source. Heterotrophic bacteria express highly specialised microbial enzymatic cascades for utilisation of these varied and complex polysaccharides as substrate for central energy-yielding metabolism.</p><p dir="ltr">To begin, genomes of <i>Rhi</i>C1 and <i>Gla</i>SL4 were annotated for the presence of putative polysaccharide utilisation loci (PUL). These are defined and coregulated genomic regions that encode carbohydrate active enzymes (CAZymes) alongside additional polysaccharide-modifying, transcription-regulatory, and transport machinery. CAZyme classes are delineated by their specific activities towards the degradation, modification, or synthesis of polysaccharides. Classes include polysaccharide lyases (PLs), glycoside hydrolases (GHs), carbohydrate esterases (CEs), glycosyl transferases (GTs), and enzymes with auxiliary activities (AA). Annotation of these enzyme classes, their respective subfamilies, and additional protein encoding genes within the defined PUL inform the likely degradation capacity and specificity of a PUL.</p><p dir="ltr">One bioinformatically validated enzymatic cascade was identified in both <i>Gla</i>SL4 and <i>Rhi</i>C1, each containing signatures of marine polysaccharide utilisation. The predicted metabolic activity of <i>Rhi</i>C1 against seaweed cell wall polysaccharides was restricted to a truncated PUL encoding three localised CAZymes (<i>Rhi</i>GH28, <i>Rhi</i>GH105, and <i>Rhi</i>PL15) and a distal <i>Rhi</i>PL12. Putative activities of these GH and PL families have not been reported to work in concert, revealing a seemingly novel putative enzymatic cascade. This complement of CAZyme machinery was biochemically investigated for their specificity and contribution to the metabolic profile of <i>Rhi</i>C1. <i>Rhi</i>GH28 was characterised as an exopolygalacturonase, contributing to the degradation of pectin species, and broadly confirming pectinolytic activity at its native locus. Putative activities of CAZymes encoded in this PUL, as well as localised transportation and transcription machinery, established a likely preference of <i>Rhi</i>C1 for substrates sourced from the wider microbiome environment, revealing the polysaccharide utilisation capacity of a microbial genus that is presently understudied in the marine environment.</p><p dir="ltr">By contrast, an expansive locus was annotated from the genome of <i>Gla</i>SL4, equipped with 19 CAZymes and eight sulfatases. The collective enzymatic potential in this PUL indicates complete degradation of the seaweed cell wall polysaccharide ulvan. This PUL presents comprehensive enzymology for the modification and degradation of ulvan, which facilitates the optimisation of ulvan’s existing bioactive properties as a sulfated, uronic acid-containing polysaccharide. Enzymes selected from this locus for biochemical investigation (<i>Gla</i>PL25a, <i>Gla</i>GH105, <i>Gla</i>S1_NC, and <i>Gla</i>S1_25) represent critical initiating steps in the cascade of ulvan degradation, the sequence of which had not been previously biochemically validated in concert. This contributes to our understanding of microbial utilisation of this complex and significant cell wall polysaccharide. Comparison between the <i>Rhi</i>C1 pPUL and the <i>Gla</i>SL4 ulvan PUL (uPUL) illustrates the complexity of marine polysaccharide utilisation and differentiation in heterotrophic polysaccharide metabolism.</p><p dir="ltr">Together, CAZymes characterised in this study enact crucial activities in seaweed cell wall polysaccharide utilisation by <i>Rhi</i>C1 and <i>Gla</i>SL4. These findings contribute not only to an improved understanding of fundamental glycan utilisation and carbon flux in the marine environment, but also the development of bespoke oligosaccharides as bioactive candidates.</p>