A Novel Enzyme Portfolio for Red Algal Polysaccharide Degradation in the Marine Bacterium Paraglaciecola hydrolytica S66T Encoded in a Sizeable Polysaccharide Utilization Locus

Abstract

Marine microbes are a rich source of enzymes for the degradation of diverse polysaccharides. Paraglaciecola hydrolytica S66T is a marine bacterium capable of hydrolyzing polysaccharides found in the cell wall of red macroalgae. In this study, we applied an approach combining genomic mining with functional analysis to uncover the potential of this bacterium to produce enzymes for the hydrolysis of complex marine polysaccharides. A special feature of P. hydrolytica S66T is the presence of a large genomic region harboring an array of carbohydrate-active enzymes (CAZymes) notably agarases and carrageenases. Based on a first functional characterization combined with a comparative sequence analysis, we confirmed the enzymatic activities of several enzymes required for red algal polysaccharide degradation by the bacterium. In particular, we report for the first time, the discovery of novel enzyme activities targeting furcellaran, a hybrid carrageenan containing both β-carrageenan and κ/β-carrageenan motifs. Some of these enzymes represent a new subfamily within the CAZy classification. From the combined analyses, we propose models for the complete degradation of agar and κ/β-type carrageenan by P. hydrolytica S66T. The novel enzymes described here may find value in new bio-based industries and advance our understanding of the mechanisms responsible for recycling of red algal polysaccharides in marine ecosystems.

A Multifunctional Polysaccharide Utilization Gene Cluster in Colwellia echini Encodes Enzymes for the Complete Degradation of κ-Carrageenan, ι-Carrageenan, and Hybrid β/κ-Carrageenan

“…Here, we demonstrate that C. echini harbors two large multifunctional polysaccharide utilization loci (PULs) encoding genes for the total catabolism of not only agar and -carrageenan but also -carrageenan and the hybrid ␤/-carrageenan furcellaran. Furthermore, we show that furcellaran degradation by C. echini is catalyzed by enzymes belonging to a new GH16_13 subfamily that are similar to those previously reported for another marine bacterium, Paraglaciecola hydrolytica S66 T (10,11). Using transcriptomics, in silico analyses, and recombinant enzyme technology, we provide more information about the reaction mechanism of GH16_13 furcellarases and characterize the oligosaccharide products that they release.…”

“…1). TonB-dependent receptors, previously proposed to be analogous to the Bacteroidetes detection system (10,19,20), were located in both gene clusters (Ce2863 and Ce345; Fig. 1).…”

“…2). Enzymes Ce367, Ce385, and Ce387 grouped with the newly characterized GH16_13 furcellaranases Ph1656, Ph1663, and Ph1675 from P. hydrolytica S66 T (10). Ce387 showed 57% identity to Ph1656 and 46% identity to Ph1675, whereas Ce367 and Ce385 displayed less than 20% identity to the furcellaranases from P. hydrolytica S66 T .…”

“…7). However, a BLASTp search revealed that sequences similar to those of Ce338 and Ce390 were discovered in several marine bacteria, including P. hydrolytica S66 T (10). Biochemical analysis showed that recombinant Ce390 hydrolyzed neocarrao- (Table S2), the level of identity between GH42 (and GH160) enzymes and Colwellia enzymes Ce338 and Ce390 was low.…”

“…In order for neocarrabiose to enter the central metabolism, this disaccharide must be hydrolyzed further to galactose and D-AHG (3,6-anhydro-D-galactose). Ficko-Blean et al (20) showed that Z. galactanivorans encodes GH127 and GH129 enzymes capable of hydrolysing D-AHG from neocarraoligosaccharides, and Schultz-Johansen et al (10) reported that a GH127 enzyme similar to those of Z. galactanivorans was found in P. hydrolytica S66 T . A search in the C. echini A3 T genome sequence for GH127 and GH129 enzymes produced negative results.…”