Biochemical Reconstruction of a Metabolic Pathway from a Marine Bacterium Reveals Its Mechanism of Pectin Depolymerization

Abstract

Pectin is a complex uronic acid-containing polysaccharide typically found in plant cell walls, though forms of pectin are also found in marine diatoms and seagrasses. Genetic loci that target pectin have recently been identified in two phyla of marine bacteria. These loci appear to encode a pectin saccharification pathway that is distinct from the canonical pathway typically associated with phytopathogenic terrestrial bacteria. However, very few components of the marine pectin metabolism pathway have been experimentally validated. Here, we biochemically reconstructed the pectin saccharification pathway from a marine Pseudoalteromonas sp. in vitro and show that it results in the production of galacturonate and the key metabolic intermediate 5-keto-4-deoxyuronate (DKI). We demonstrate the sequential de-esterification and depolymerization of pectin into oligosaccharides and the synergistic action of glycoside hydrolases (GHs) to fully degrade these oligosaccharides into monosaccharides. Furthermore, we show that this pathway relies on enzymes belonging to GH family 105 to carry out the equivalent chemistry afforded by an exolytic polysaccharide lyase (PL) and KdgF in the canonical pectin pathway. Finally, we synthesize our findings into a model of marine pectin degradation and compare it with the canonical pathway. Our results underline the shifting view of pectin as a solely terrestrial polysaccharide and highlight the importance of marine pectin as a carbon source for suitably adapted marine heterotrophs. This alternate pathway has the potential to be exploited in the growing field of biofuel production from plant waste. IMPORTANCE Marine polysaccharides, found in the cell walls of seaweeds and other marine macrophytes, represent a vast sink of photosynthetically fixed carbon. As such, their breakdown by marine microbes contributes significantly to global carbon cycling. Pectin is an abundant polysaccharide found in the cell walls of terrestrial plants, but it has recently been reported that some marine bacteria possess the genetic capacity to degrade it. In this study, we biochemically characterized seven key enzymes from a marine bacterium that, together, fully degrade the backbone of pectin into its constituent monosaccharides. Our findings highlight the importance of pectin as a marine carbon source available to bacteria that possess this pathway. The characterized enzymes also have the potential to be utilized in the production of biofuels from plant waste.

Sweet and magnetic: Succession and CAZyme expression of marine bacterial communities encountering a mix of alginate and pectin particles

“…These results corroborate that pectin was not a prime bacterial substrate, although pectinolytic bacteria occur in diverse marine habitats [70,87,88] and pectinous substrates are exuded by Helgoland macroalgae [10]. Altogether, the evidence suggests that PecP primarily served as colonization scaffolds.…”

“…Alternatively, despite lower transcript abundances, a GH105 gene in another PUL (Fig. 7B) might mediate the exolysis of polymeric pectin, allowing complete saccharification without pectate lyases [87]. The majority of CAZymes in MAG21 possesses homologs in Catenovulum spp.…”

Genes for degradation and utilization of uronic acid-containing polysaccharides of a marine bacteriumCatenovulumsp. CCB-QB4

“…One is that the disaccharides are degraded by oligogalacturonate lyase belonging to family PL22, and then the product, Δ-4,5-unsaturated galacturonate, is linearized into DKI by KdgF ( Hobbs et al, 2016 ). The other is that unsaturated galacturonyl hydrolases belonging to GH105 degrades the disaccharides and directly releases DKI ( Hobbs et al, 2019 ). In the QB4 genome, no PL22 enzymes were found in the genome.…”

Insights into the κ/ι-carrageenan metabolism pathway of some marine Pseudoalteromonas species

“…Growth of Pseudoalteromonas isolates on ι-carrageenan and carrageenan oligosaccharides was assessed at 25 °C in a SpectraMax M5 plate reader at 600 nm essentially as previously described 50 . Quadruplicate overnight cultures of each strain were grown in Zobell broth from individual colonies, washed with minimal marine medium (MMM) 51 and resuspended in MMM containing no carbon source.…”