Engineering of the Flavone Rutinoside Biosynthetic Pathway Enables Production of Diosmin, a Venoactive Compound in <scp> <i>Solanum lycopersicum</i> </scp> and <scp> <i>Nicotiana tabacum</i> </scp>

Diosmin is often used as a dietary supplement, and it is sometimes used to make phlebotropic drugs for venous diseases. Traditionally, diosmin is obtained by the dehydrogenation of the flavanone glycoside hesperidin through chemical semisynthesis. In this study, we identified and characterised a flavonoid-O-methyltransferase (F4'OMT) and two glucosyltransferases (F7GlcTs) and completed the biosynthetic pathway of diosmin and its analogue, linarin and isorhoifolin, in combination with the previously found flavone synthase (FNSII) and rhamnosyltransferase (RhaT). We developed an isocaudarner-based gene stacking strategy to integrate FNS, OMT, GlcT and RhaT into a multi-gene vector that was transformed into Solanum lycopersicum cv. 'MicroTom' and Nicotiana tabacum. The introduction of the flavone pathway enabled the production of diosmin at 474 ng/g DW in the peel of transgenic tomato and 20.5 ng/g DW in the leaves of tobacco. In addition, linarin with a 299.5 ng/g level and isorhoifolin with a 2.8 ng/g level can be found in the leaves of tobacco. How the endogenous enzymes and metabolic flux affected the production of the specialised compounds was discussed. Our results represent the first attempt in the heterologous biosynthesis of O-methylated and rhamnosylated flavone di-glucosides in flavonol-producing plants.