A Push-and-Pull metabolic strategy drives constitutive paramylon hyper-accumulation in a self-harvesting Euglena gracilis mutant

The commercial production of paramylon, a high-value beta-1,3-glucan from Euglena gracilis, is currently hindered by the inherent trade-off between cell growth and product accumulation, as well as high harvesting costs. Here, we overcame these bottlenecks by establishing a high-yield mutant strain, EGMS, using ethyl methanesulfonate (EMS) mutagenesis coupled with a targeted "dark-heterotrophic" morphological screening strategy. Under heterotrophic conditions, EGMS exhibited a "constitutive" high-yield phenotype, successfully decoupling biomass accumulation from nutrient starvation. Uniquely, the mutant adopted a spherical, high-density morphology due to massive paramylon granule accumulation acting as an intracellular ballast. This trait enabled rapid gravitational sedimentation (increasing efficiency by ~119%), effectively resolving the harvesting challenge without chemical flocculation. Integrated transcriptomic and metabolomic analyses revealed a sophisticated "Push-and-Pull" regulatory mechanism driving this phenotype. The "Push" force was generated by the robust activation of central carbon metabolism and paramylon synthase genes (UGPase, GSL), ensuring a continuous supply of the precursor UDP-glucose. Complementing this, a decisive "Pull" (or blockade) was enforced by the deep repression of competing lipid elongation (KCS) and sulfur assimilation (CysK) pathways, effectively channeling carbon flux into the paramylon sink. This precise metabolic redirection appears to be orchestrated by a hierarchically rewired bZIP-MYB transcriptional module. Collectively, EGMS represents a robust, easy-harvesting industrial chassis for cost-effective paramylon production, offering novel insights into carbon flux regulation in heterotrophic microalgae.