Abstract
Volvariella volvacea, a fungal species of Volvariella within the Pluteaceae family, is predominantly cultivated in southern China. Polysaccharides, the primary bioactive constituents of V. volvacea, exhibit diverse pharmacological activities. However, current cultivation practices face challenges due to the genetic heterogeneity of strains, leading to inconsistent content and compositional variability of polysaccharides and other functional components. ARTP, denoting atmospheric and room-temperature plasma, is a technology capable of generating plasma jets at ambient pressure with temperatures ranging from 25 to 40 °C. These jets feature high concentrations of highly reactive species, including but not limited to excited-state helium atoms, oxygen atoms, nitrogen atoms, and OH radicals. This study aims to develop high-yielding exopolysaccharide (EPS) strains through integrated ARTP mutagenesis and genome shuffling, thereby overcoming current cultivation bottlenecks. ARTP mutagenesis and genome shuffling significantly boosted EPS production in V. volvacea. ARTP generated nine stable mutants with >20% higher EPS yields. Subsequent genome shuffling (three rounds of protoplast fusion) produced the hybrid strain SL212, which achieved 46.85 g/L of EPS, an 111.67% increase over that of the parent strain under identical conditions. Metabolomics and transcriptomics analyses revealed that differential metabolites and genes were mainly enriched in galactose metabolism, ABC transporter pathways, and the tricarboxylic acid cycle. These pathways enhance monosaccharide biosynthesis and generate ATP, providing both precursors and energy for polysaccharide polymerization, thereby driving EPS overproduction. Preliminary mechanistic analysis identified the key contributing factors driving the elevated polysaccharide biosynthesis.