title: Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures.
abstract: Grapevine stilbenes, particularly trans-resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering-based strategy to produce resveratrol derivatives using resveratrol-converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O-methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra- and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild-type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.
authors.affiliation: Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Faculty of Science, University of Alicante, Alicante, Spain.
authors.forename: Ascensión
authors.initials: A
authors.lastname: Martínez-Márquez
authors.affiliation: Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Faculty of Science, University of Alicante, Alicante, Spain.|Biotechnology and Molecular Biology Group, Quevedo State Technical University, Quevedo, Ecuador.
authors.forename: Jaime A
authors.initials: JA
authors.lastname: Morante-Carriel
authors.affiliation: Laboratory of Plant Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain.
authors.forename: Karla
authors.initials: K
authors.lastname: Ramírez-Estrada
authors.affiliation: Laboratory of Plant Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain.
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Cusidó
authors.affiliation: Laboratory of Plant Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Palazon
authors.affiliation: Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Faculty of Science, University of Alicante, Alicante, Spain.
authors.forename: Roque
authors.initials: R
authors.lastname: Bru-Martínez
mesh_terms: D018929:Cell Culture Techniques; D018506:Gene Expression Regulation, Plant; D060847:Metabolic Engineering; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D000077185:Resveratrol; D013267:Stilbenes / Q000737:chemistry / Q000378:metabolism*; D027843:Vitis / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*
chemical_list: D013267:Stilbenes; C107773:pterostilbene; C041525:3,3',4,5'-tetrahydroxystilbene; D000077185:Resveratrol
keywords: Vitis vinifera; grapevine cell culture; metabolic engineering; piceatannol; pterostilbene; resveratrol