Multigene synergism increases the isoflavone and proanthocyanidin contents of Medicago truncatula

Isoflavones and proanthocyanidins (PAs), which are flavonoid derivatives, possess many health benefits and play important roles in forage‐based livestock production. However, the foliage of Medicago species accumulates limited levels of both isoflavones and PAs. In this study, biosynthesis of isoflavone and PA in Medicago truncatula was enhanced via synergy between soya bean isoflavone synthase (IFS1); two upstream enzymes, chalcone synthase (CHS) and chalcone isomerase (CHI); and the endogenous flavanone 3‐hydroxylase (F3H). Constitutive expression of GmIFS1 alone resulted in ectopic accumulation of the isoflavone daidzein and large increases in the levels of the isoflavones formononetin, genistein and biochanin A in the leaves. Furthermore, coexpression of GmIFS1 with GmCHS7 and GmCHI1A generally increased the available flux to flavonoid biosynthesis and resulted in elevated isoflavone, flavone and PA contents. In addition, down‐regulation of MtF3H combined with coexpression of GmIFS1, GmCHS7 and GmCHI1A led to the highest isoflavone levels (up to 2 μmol/g fresh weight in total). Taken together, our results demonstrate that multigene synergism is a powerful means to enhance the biosynthesis of particular flavonoids and can be more broadly applied to the metabolic engineering of forage species.