Abstract
The potential of pharmacologically active secondary plant metabolites is limited by the low yield from often rare plants, and the lack of economically feasible chemical synthesis of these complex compounds. Plant cell fermentation offers an alternative strategy to overcome these constraints. However, the efficiency of this approach is limited by intracellular sequestration of the products, such that continuous bioprocessing is not possible. As a precondition for such a, more attractive, continuous process, it is of great importance to stimulate the export of the product into the medium without impairing viability and, thus, the productivity of the cells. Using nicotine alkaloids of tobacco as a case study, an alternative strategy is explored, where nanosecond pulsed electric fields (nsPEFs) are applied for the efficient downstream recovery of the products. To maintain cell viability and allow for the further use of biomass, cells were exposed to strong (1–20 kV·cm−1), but very short (10–100 ns) electric pulses, which leads to a temporary permeabilisation of cell membranes. Using two transgenic cell lines, where two key genes involved in the metabolism of the anti-Alzheimer compound nornicotine were overexpressed, we could show that this nsPEF treatment improved the partitioning of some nicotine alkaloids to the culture medium without impairing viability, nor the synthesis of alkaloids. However, this release was only partial and did not work for nornicotine. Thus, nsPEFs produced a fractionation of alkaloids. We explain this electrofractionation by a working model considering the differential intracellular compartmentalization of nicotineic alkaloids.
Highlights
Handling Editor: Handling Editor: Jaideep MathurElectronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Molecular Cell Biology, Botanical Institute, Karlsruhe Institute of Technology, Karlsruhe, GermanyInstitute for Pulsed Power and Microwave Technology (IHM), Karlsruhe Institute of Technology, Karlsruhe, GermanyPlants are endowed with an impressive ability to produce metabolites, especially secondary compounds (Goossens et al 2003; Anarat-Cappillino and Sattely 2014)
We explored the use of nanosecond pulsed electric fields (nsPEFs) for plant cell fermentation of a pharmaceutically interesting compound, nornicotine, using metabolic engineering in transgenic strains of BY-2 (Nicotiana tabacum L. cv Bright Yellow 2), a model cell line for plant research (Nagata et al 1992)
A set of different pulsing indices were tested for their ability to release nicotineic alkaloids into the medium, while preserving viability (Table 1)
Summary
The bark of up to twelve individual Taxus brevifolia trees was needed to provide the taxol required to cure one ovarian cancer patient (Joyce 1993) Since this approach is not sustainable, parallel efforts have been launched to find alternative ways for the production of these compounds, rather than relying solely on natural sources (Bourgaud et al 2001). Secretion of valuable metabolites to the culture medium can significantly decrease downstream processing costs (Wilson and Roberts 2012), but is often limited by the pecto-cellulosic cell wall, and more importantly, by the plasma membrane, sometimes by the membranes of intracellular compartments (Georgiev et al 2009)
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