Coculture of genetically transformed roots and shoots for synthesis, translocation, and biotransformation of secondary metabolites

Abstract

Genetically transformed shooty teratomas of Atropa belladonna and a Duboisia leichhardtii X D. myoporoides hybrid were studied for biotransformation of tropane alkaloids in shake flasks and bioreactors. Although de novo synthesis of hyoscyamine and scopolamine was limited, shoots of both species were able to translocate and accumulate significant quantities of exogenous alkaloid. The maximum yield of scopolamine from hyoscyamine fed to the Duboisia hybrid shoots was 35% w/w; the yield of the scopolamine precursor, 6ph yd roxyh yoscya m i ne, was 37% w/w. Biotransformation activity was poor in A. belladonna shooty teratomas provided with exogenous hyoscyamine; however, scopolamine levels comparable with those in leaves of the whole plant accumulated in shoots fed with hairy root extract. Coculture of A. belladonna shooty teratomas and hairy roots in the same hormone-free medium was investigated as a means of providing a continuous source of hyoscyamine for conversion to scopolamine. Of the biotransformation systems tested with A. belladonna, coculture produced the highest levels of scopolamine and the highest scopolamine: hyoscyamine ratios. Cocultured shoots accumulated up to 0.84 m g g-' dry weight scopolamine, or 3-1 1 times the average concentrations found in leaves of the whole plant. The scopolamine : hyoscyamine ratio in coculture ranged from 0.07 to 1.9, a significant improvement over levels of 0-0.03 normally found in A. belladonna hairy roots. Addition of Pluronic F-68 or copper sulfate t o the medium and variation in initial medium pH did not improve hyoscyamine release from hairy roots. Scopolamine levels were increased using 1 p M copper sulfate or initial medium pH between 6.0 and 8.0; however, results from elicitation of hairy roots could not match the beneficial effect on scopolamine synthesis of root-shoot coculture. Addition of 0.001-1.0% (w/v) Pluronic F-68 to the roots reduced hyoscyamine release but postponed necrosis in the root tissue for up t o 60 d. 0 1996 John Wiley & Sons, Inc.

Combination of Plant Metabolic Modules Yields Synthetic Synergies

“…Co-culture of root and shoot has beed used previously to improve production of tropane alkaloids in Atropa belladonna and a Duboisia leichhardtii × D . myoporoides hybrid plants [51]. The significant increase in nornicotine production by Ntom CYP82E4 when co-cultivated with Ntab MPO1 cells demonstrated that the physical presence of cells was superior to just feeding the conditioned medium collected from the same cells (Fig 8).…”

Production of Podophyllotoxin Using Cross‐Species Coculture of Linum flavum Hairy Roots and Podophyllum hexandrum Cell Suspensions

“…This study represents an extension of previous work in which single‐ and cross‐species coculture of hairy roots and shooty teratomas was used to enhance the production of scopolamine in vitro (25, 26). Because neither hairy roots nor shooty teratomas require exogenous growth regulators, the choice of medium for this type of coculture is relatively straightforward.…”

Influence of inoculum morphology on growth of Atropa belladonna hairy roots and production of tropane alkaloids

“…The procedure used for analysis of tropane alkaloids was based on that described by Subroto et al (1996). Ion-pair HPLC was carried out using a reverse-phase column (Alltima C 8, 5 ~tm, 250 mmx 4.6 mm; Alltech, USA) operated at ambient temperature with UV detection at 205 nm.…”