Ectopic expression of a deletion variant of cystathionine gamma-synthase 2 (GmCGS2) in soybean increases expression of genes encoding sulfur amino acid-rich proteins

Soybean Met and Cys contents are sub-optimal for livestock feed. Previously, cystathionine gamma-synthase (CGS) overexpression in plants increased these amino acids but also resulted in severe deleterious phenotypes. The Met-rich and Cys-rich proteins that contributed to the improved nutritional value were not identified. A deletion variant of GmCGS2 was ectopically expressed in soybean with a diverse range of expression levels and patterns. A combination of a soybean ubiquitin promoter and an in-frame deletion in GmCGS2, from K66 to S143, resulted in increased seed Met and Cys contents without incurring a yield penalty in a ten-location field trial. Transgenic events of this construct had increases of 41 to 114%, 10 to 12%, and 4.7 to 7.3% in free Met, total Cys, and total Met, respectively. RNAseq analysis of developing seed revealed increased transcript for three Cys-rich Bowman-Birk protease inhibitor (BBI) genes, and for two Met-rich 2 S albumin genes, with little effect apparent on genes of primary sulfur metabolism. Trypsin-agarose chromatography, protease inhibition assays, and mass spectrometry analysis revealed increased BBI and 2 S albumin proteins. The additional BBI protein was determined to be 1.5 to 1.8% of the protein in transgenic seed, sufficient to account for the increased Cys content. The observation that overexpressing an enzyme involved in Met biosynthesis that used Cys as a substrate still increased total Cys more than total Met suggested a lack of native Met-rich soybean proteins capable of incorporating the additional free Met. This study demonstrated that CGS can be manipulated to increase soybean seed total sulfur amino acid content without incurring a yield penalty, identified sulfur amino acid-rich proteins that contributed to the compositional improvement, and provided rationale for why engineering high-Met soybean proteins may be warranted.