Combining pathway engineering and precursor enrichment to build a multi-trait soybean platform for aquaculture feed...

Oilseed crops offer a scalable aquaculture feed production platform for generating oils with long-chain ω-3 polyunsaturated fatty acids and high-value antioxidants, but metabolic flux through heterologous pathways is often constrained by precursor availability and endogenous regulatory limits. Here we engineered soybean seeds to co-produce eicosapentaenoic acid (EPA), astaxanthin, and tocotrienols by introducing nine microbial and plant enzymes spanning fatty acid desaturation/elongation, ketocarotenoid synthesis, and vitamin E tocochromanol biosynthesis. Transgenic events accumulated EPA up to ∼3.8% of total seed fatty acids, but high concentrations of undesirable ω-6 intermediates and weak expression of Δ12 and Δ17 desaturases suggested substrate limitation as a major flux bottleneck. These flux limitations reflect inherent metabolic trade-offs in complex pathway engineering, prompting the crossing of the engineered line with a high-α-linolenic acid (ALA) soybean to expand the ω-3 precursor pool. This metabolic breeding strategy increased EPA to ∼14% of total fatty acids, a fourfold improvement, and restored several physiological defects, including poor germination and reduced oil deposition. The crossed line retained astaxanthin (13 μg g^-1) and tocotrienols (1300 μg g^-1), demonstrating stable multi-trait accumulation. Metabolic, hormonal, and transcriptomic analyses indicate that competition for carbon and geranylgeranyl diphosphate contributed to the oil and ABA reductions observed in the original line. These results show that precursor-pool expansion can resolve major flux constraints in engineered seeds and illustrate a combined synthetic-pathway and breeding approach for constructing multi-trait oilseed platforms for sustainable aquafeed applications.