Re-Engineering Soybean Protein Quality: Toward Low Trypsin Inhibitor Soybean Using Classical Breeding and Genome Editing to Target KTI and BBI

Soybean seeds have long been regarded as “storehouses of high-quality proteins”. The breakdown of dietary proteins by digestive proteases is essential for achieving adequate protein digestibility in animals and humans. However, plants have evolved a diverse array of protease inhibitors that regulate or restrict protease activity. In soybean, these inhibitors are concentrated primarily within the 2S protein fraction. Trypsin inhibitors (TIs) of Kunitz trypsin inhibitor (KTI) and Bowman–Birk inhibitor (BBI) are the most impactful due to their strong anti-tryptic activity, which interferes with digestive proteases in humans and animals. Elevated TI levels render raw soybeans unsuitable for direct food or feed use unless thermal or processing inactivation treatments are applied. Elimination or reduction in KTI and BBI using classical and biotechnology-based breeding efforts is a promising strategy. Soybean germplasm harboring BBI null alleles has not been reported. Breeding only for low or null KTI content in soybean would not be sufficient for practical applications. Hybridizing IT105782 × PI 547656 and using the reported Kompetitive Allele-Specific PCR (KASP) markers represents an effective classical breeding strategy. Simultaneous CRISPR/Cas9-mediated knockout of key KTI and BBI genes is expected to enable the development of soybean lines with substantially reduced TI levels, an outcome that cannot be readily achieved through classical introgression of null alleles, as naturally occurring null BBI alleles have not yet been identified. Moreover, this approach avoids the linkage drag associated with donor-derived null KTI alleles. However, this approach remains challenging due to functional redundancy and compensatory effects among KTI and BBI family members, extensive sequence homology among KTI and BBI genes that complicates the minimization of off-target effects, and the genotype dependency of Agrobacterium-mediated soybean transformation. Microtiter plate AACCI/AOCS could be one practical option for measuring TIA in breeding programs in terms of precision. Potential trade-offs associated with reduced trypsin inhibitor levels, including possible effects on plant defense and stress resistance, should be investigated in future studies, as these aspects have received little attention in previous research.