Improved production of the antidiabetic metabolite montbretin A in Nicotiana benthamiana: discovery, characterization, and use of Crocosmia shikimate shunt genes.

Abstract

The plant-specialized metabolite montbretin A (MbA) is being developed as a new treatment option for type-2 diabetes, which is among the ten leading causes of premature death and disability worldwide. MbA is a complex acylated flavonoid glycoside produced in small amounts in below-ground organs of the perennial plant Montbretia (Crocosmia × crocosmiiflora). The lack of a scalable production system limits the development and potential application of MbA as a pharmaceutical or nutraceutical. Previous efforts to reconstruct montbretin biosynthesis in Nicotiana benthamiana (Nb) resulted in low yields of MbA and higher levels of montbretin B (MbB) and montbretin C (MbC). MbA, MbB, and MbC are nearly identical metabolites differing only in their acyl moieties, derived from caffeoyl-CoA, coumaroyl-CoA, and feruloyl-CoA, respectively. In contrast to MbA, MbB and MbC are not pharmaceutically active. To utilize the montbretia caffeoyl-CoA biosynthesis for improved MbA engineering in Nb, we cloned and characterized enzymes of the shikimate shunt of the general phenylpropanoid pathway, specifically hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (CcHCT), p-coumaroylshikimate 3'-hydroxylase (CcC3'H), and caffeoylshikimate esterase (CcCSE). Gene expression patterns suggest that CcCSE enables the predominant formation of MbA, relative to MbB and MbC, in montbretia. This observation is supported by results from in vitro characterization of CcCSE and reconstruction of the shikimate shunt in yeast. Using CcHCT together with montbretin biosynthetic genes in multigene constructs resulted in a 30-fold increase of MbA in Nb. This work advances our understanding of the phenylpropanoid pathway and features a critical step towards improved MbA production in bioengineered Nb.