Delving into Enzyme-Encoding Genes in Metagenome Assembled Genomes from Artisanal Dairies Producing Raw Ewe Milk PDO Cheeses

Artisanal raw milk cheeses harbour complex microbial communities that drive cheese making and shape sensory quality. Previous work on Idiazabal cheese identified rennet as a major microbial source, although all reservoirs contributed to varying degrees. However, their impact in terms of enzyme-encoding genes related to technological quality of cheese remained unexplored. Building on that, this study draws on metagenome-assembled genomes (MAGs) from cheeses and dairy environments to comprehensively identify enzyme-encoding genes involved in key biochemical processes. In cheese MAGs (Lacticaseibacillus paracasei), protease-encoding genes were dominated by ATP-dependent metalloproteases (M41), carbohydrate-active enzyme-encoding genes (CAZymes) by glycoside hydrolases (GH) and glycosyltransferases (GT), while esterase, lipase, and related-enzyme-encoding genes were restricted to sparse ‘GDXG’, type-B and esterase D families. Dairy environments emerged as major reservoirs of enzyme-encoding genes, with notable differences among sample types (p ≤ 0.001). The richest sources of protease-encoding genes were grass (610 genes), linked primarily to Pantoea agglomerans, and rennet (318), mainly related to Basfia sp. and Moraxella sp., dominated by metalloproteases (M23, M38) and serine proteases (S15). The largest reservoirs of CAZyme-encoding genes were food contact surfaces (1550), associated mainly with Salinisphaera sp. and Dietzia sp., and rennet (1505), related to, e.g., Bacteroides pyogenes, Alloprevotella sp., and Lentilactobacillus buchneri. Food contact surfaces were also the richest source of esterase, lipase and related-enzyme-encoding genes (1209), mainly linked to Dietzia sp., Corynebacterium sp., and Brevibacterium aurantiacum. Similarly, aroma-related enzyme-encoding genes (e.g., oppA, pepA, GH13, esterase D) were consistently detected in environmental matrices. These results provide novel insights into dairy microbiomes as functional reservoirs of aroma precursors, revealing their relevance for artisanal PDO cheese production and future biotechnological applications.