Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Abstract

SummaryGrapevine 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 (Vv ROMT) or human cytochrome P450 hydroxylase 1B1 (Hs CYP1B1) 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 Vv ROMT or Hs CYP1B1. Grapevine cell cultures transformed with Vv ROMT produced pterostilbene, which was detected in both intra‐ and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with Hs CYP1B1 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.

Bioconversion of stilbenes in genetically engineered root and cell cultures of tobacco

“…However, the positive effect of MBCD on the release of reaction products to the culture medium could negatively affect the production of the system if they are less stable in the medium. This may be the case with t -Pt or piceid, which were absent in grapevine cell cultures even after elicitation with MeJA and MBCD16.…”

“…Recently, in a similar approach, Martinez-Marquez et al 16. reported a 200-fold enhancement of t -Pn production in grapevine cell cultures by heterologous expression of the Hs CYP1B1 gene, and the presence of t -Pt in transgenic cell lines overexpressing the Vv ROMT gene, when both cultures were elicited with MeJA and MBCD.…”

“…For stilbenoid extraction from cells, four parts of 100% methanol were added per g of fresh weight, with stirring at 115 rpm for 24 h. The methanol extract was filtered and brought to dryness16. All samples were resuspended in 1 mL of 80% methanol and sonicated for 30 min and filtered through a 0.22 μm PVDF filter just before analysis.…”

“…The use of strong promotors to overexpress genes involved in biosynthetic pathway bottlenecks is currently a common strategy for improving the production of target compounds in engineered biological systems15. In this scenario, Martinez-Marquez et al 16. recently showed that constitutive expression of resveratrol O-methyltransferase in Vitis vinifera led to the production of t -Pt, and the heterologous expression of the human cytochrome P450 hydroxylase 1B1 ( Hs CYP1B1) increased t -Pn accumulation in elicited grapevine cell cultures.…”

Silybum marianum cell cultures stably transformed with Vitis vinifera stilbene synthase accumulate t‐resveratrol in the extracellular medium after elicitation with methyl jasmonate or methylated β‐cyclodextrins

“…A binary vector pJCV52‐STS3 was built following the protocol described in . The VvSTS3 gene was cloned into the pJCV52 vector (Laboratory of Plant Systems Biology; Ghent University, Belgium) under the control of CaMV35S promoter using the Gateway cloning system (Invitrogen, Life Technologies, NY, USA).…”