Targeted Reduction of Specific Antinutritional and Allergen-Associated Proteins to Advance Soybean Seed Quality

Soybean (Glycine max L.) is a major global source of protein and oil, yet its use in food and feed is constrained by antinutritional factors (ANFs) and allergenic seed proteins. To improve seed nutritional quality, we developed double mutant soybean lines (HBK-WF and HBK-PF) by crossing E16, which lacks the Kunitz trypsin inhibitors KTI-1 and KTI-3, with Lee 2, which is deficient in the α subunit of β conglycinin, a prominent allergen-associated storage protein. One- and two-dimensional gel electrophoresis and immunoblot analyses confirmed the complete absence of these target proteins in both experimental lines. The double mutants exhibited ~50% lower trypsin inhibitor activity relative to wild-type soybean. Tandem mass tag (TMT)-based proteomic analysis quantified 1034 proteins across parental and derived genotypes and revealed extensive proteomic remodeling. Seed composition profiling demonstrated substantial genotypic variation. Lee 2 displayed the highest total protein concentration (44%), while the double mutants exhibited intermediate to elevated levels (39–40%). Oil content in the double mutants (18.4–18.5%) was comparable to E16 and slightly lower than the reference cultivar Maverick. Amino acid profiling further showed that Lee 2 accumulated the highest levels of all measured amino acids, particularly sulfur-containing amino acids, whereas the double mutants displayed intermediate concentrations consistent with additive inheritance. Collectively, these results demonstrate that targeted removal of specific ANFs and an allergen-associated storage protein can improve soybean nutritional quality without compromising seed composition, providing promising germplasm for enhancing the value of soybean-derived food and feed products.