Detoxifying and depolymerizing microorganisms reveal intertwined guild collaborations in the gut microbiome of the generalist macro-algivorous fish Kyphosus cinerascens

The biotransformation of macroalgal biomass represents a major catabolic challenge due to its structurally diverse polysaccharides and inhibitory polyphenols. Unlike terrestrial lignocellulosic substrates, macroalgal polysaccharides contain multiple monomer types, branching patterns, and sulfation states. Additionally, toxic macroalgal polyphenols have been shown to inhibit both microbial growth and their catalytic enzymes. While herbivorous fishes have evolved specialized gut microbiota to process these substrates, the enzymatic pathways remain poorly characterized, with few experimentally validated polysaccharide utilization loci or biochemically defined marine sulfatases, and limited understanding of polyphenol degradation. Here, we developed in vitro microcosms, based on the gut microbiome of the generalist macro-algivorous fish Kyphosus cinerascens , to temporally resolve the activity of the microbial guilds involved in macroalgal polysaccharide and polyphenol transformation. First, parallel cDNA/DNA amplicon sequencing was employed to distinguish the natural active fraction from transient gut microbiome taxa that became inactive/dead after their ingestion. Four medium combinations were able to propagate between 96% and 99% of the active hindgut microbial families, reproducing the cooperative degradation dynamics observed in vivo . Metagenomic and metatranscriptomic profiling of these four optimized in vitro microcosms served as models to assess the stepwise functional successions occurring in the natural gut microbiome. Early Gammaproteobacteria expressed enzymes linked to polyphenol detoxification and alginate degradation, followed by Bacillota, Bacteroidota, and Verrucomicrobiota guilds targeting more recalcitrant sulfated polysaccharides and polyphenols. Together, these results identified temporal and taxonomic coordination as key features of macroalgal biomass deconstruction, providing an experimentally tractable model for discovering novel carbohydrate-active enzymes and elucidating poorly understood pathways of marine polyphenol degradation.