Rosa hybrida orcinol O-methyl transferase-mediated production of pterostilbene in metabolically engineered grapevine cell cultures

Abstract

Stilbenes are naturally scarce high-added-value plant compounds with chemopreventive, pharmacological and cosmetic properties. Bioproduction strategies include engineering the metabolisms of bacterial, fungal and plant cell systems. Strikingly, one of the most effective strategies consists in the elicitation of wild grapevine cell cultures, which leads to vast stilbene resveratrol accumulation in the extracellular medium. The combination of both cell culture elicitation and metabolic engineering strategies to produce resveratrol analogs proved more efficient for the hydroxylated derivative piceatannol than for the dimethylated derivative pterostilbene, for which human hydroxylase HsCYP1B1- and grapevine O-methyltransferase VvROMT-transformed cell cultures were respectively used. Rose orcinol O-methyltransferase (OOMT) displays enzymatic properties, which makes it an appealing candidate to substitute VvROMT in the combined strategy to enhance the pterostilbene production level by engineered grapevine cells upon elicitation. Here we cloned a Rosa hybrida OOMT gene, and created a genetic construction suitable for Agrobacterium-mediated plant transformation. OOMT's ability to catalyze the conversion of resveratrol into pterostilbene was first assessed in vitro using protein extracts of agroinfiltrated N. benthamiana leaves and transformed grapevine callus. The grapevine cell cultures transformed with RhOOMT produced about 16 mg/L culture of pterostilbene and reached an extracellular distribution of up to 34% of total production at the best, which is by far the highest production reported to date in a plant system. A bonus large resveratrol production of ca. 1500-3000 mg/L was simultaneously obtained. Our results demonstrate a viable successful metabolic engineering strategy to produce pterostilbene, a resveratrol analog with enhanced pharmacological properties.

Keywords: Grapevine cell culture; Metabolic engineering; Orcinol O-methyltransferase; Pterostilbene; Resveratrol; Vitis vinifera.