Saponin free yellow pea seeds by CRISPR/Cas9 enabled mutagenesis of beta-amyrin synthase

The use of yellow pea protein commercially is hindered by the presence of off-flavor metabolites known as saponins. Saponins have bitter and astringent tastes and are difficult to separate from pea proteins due to their amphiphilic nature. Saponins are glycosylated triterpenes, the triterpene backbone is derived from central carbon metabolism via beta-amyrin synthase which cyclizes 2,3-oxidosqualene. The tight control that beta-amyrin has as the branch point enzyme into saponin biosynthesis makes it an obvious target for CRISPR/Cas9 induced mutagenesis as a saponin deficient trait can be generated by a simple single gene knock-out. Therefore, we developed a CRISPR/Cas9 mutagenesis system for mutating the pea beta-amyrin synthase 1. The generated mutants had a 99% reduction in seed saponin content demonstrating the utility of CRISPR/Cas9 in rapidly producing dramatically improved pea varieties.