Seed-specific silencing of the abundantly expressed soybean Bowman-Birk protease inhibitor genes by RNAi lowers the trypsin and chymotrypsin inhibitor activities and enhances the protein digestibility

Authors: KIM, WONSEOK - University Of Missouri; KIM, SUNHYUNG - University Of Missouri; Krishnan, Hari

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/17/2025
Publication Date: 7/19/2025
Citation: Kim, W., Kim, S., Krishnan, H.B. 2025. Seed specific silencing of the abundantly expressed soybean Bown-Birk protease inhibitor genes by RNAi lowers the trypsin and chymotrypsin inhibitor activities and enhances the protein digestibility. International Journal of Molecular Sciences. 26(14). https://doi.org/10.3390/ijms26146943.
DOI: https://doi.org/10.3390/ijms26146943

Interpretive Summary: Soybean meal, with its complete amino acid profile, is an excellent protein source for livestock. However, it cannot be directly used in animal feed mixtures due to the presence of various anti-nutritional compounds that can hinder weight gain in animals. Among these compounds, protease inhibitors are particularly problematic as they interfere with the activity of digestive enzymes, specifically trypsin and chymotrypsin. Currently, no soybean cultivars have been developed that are entirely devoid of chymotrypsin inhibitor activity, primarily due to the presence of the Bowman-Birk protease inhibitor (BBI) in soybean seeds. In this study, we successfully employed RNA interference (RNAi) technology to knock out the expression of BBI genes in soybean seeds. The resulting BBI mutants exhibited a significant reduction in trypsin and chymotrypsin inhibitor activities and showed improved protein digestibility. These newly developed BBI knockout lines are likely to require minimal heat processing, leading to substantial cost savings during production. The improved digestibility underscores the potential of BBI knockout soybeans as a superior ingredient in animal feed, providing both economic and nutritional benefits to American farmers and consumers.

Technical Abstract: Soybean meal (SBM) is extensively used as a predominant protein source in animal feed. However, raw soybean cannot be directly utilized in animal feed, due to the presence of the Kunitz trypsin inhibitor (KTi) and the Bowman–Birk protease inhibitor (BBi). These antinutritional factors inhibit the digestive enzymes in animals, trypsin and chymotrypsin, resulting in poor animal performance. To inactivate the activity of protease inhibitors, SBM is subjected to heat processing, a procedure that can negatively impact the soybean protein quality. Thus, it would be beneficial to develop soybean varieties with little or no trypsin inhibitors. In this study, we report on the creation of experimental soybean lines with significantly reduced levels of Bowman–Birk protease inhibitors. RNA interference (RNAi) technology was employed to generate several transgenic soybean lines. Some of these BBi knockdown soybean lines showed significantly lower amounts of both trypsin and chymotrypsin inhibitor activities. Western blot analysis revealed the complete absence of BBi in selected RNAi-derived lines. RNA sequencing (RNAseq) analysis demonstrated a drastic reduction in the seed-specific expression of BBi genes in the transgenic soybean lines during seed development. Confocal fluorescence immunolabeling studies showed that the accumulation of BBi was drastically diminished in BBi knockdown lines compared to wild-type soybeans. The absence of BBi in the transgenic soybean did not alter the overall protein, oil, and sulfur amino acid content of the seeds compared to wild-type soybeans. The seed protein from the BBi knockdown lines were more rapidly hydrolyzed by trypsin and chymotrypsin compared to the wild type, indicating that the absence of BBi enhances protein digestibility. Our study suggests that these BBi knockdown lines could be a valuable resource in order for plant breeders to incorporate this trait into commercial soybean cultivars, potentially enabling the use of raw soybeans in animal feed.