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Secondary metabolites from plant cell cultures

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Title: Secondary metabolites from plant cell cultures


1
Secondary metabolites from plant cell cultures
  • Suggested reading Vasil ch 11, Bhojwani ch17
  • Types of secondary metabolites (SMs)
  • pharmaceuticals from plant products (e.g.,
    alkaloids, analgesics, cancer-fighting chemicals)
  • plant-derived flavors and colorings (where
    natural compounds are necessary/desirable
  • SMs made in
  • batches (small to medium-scale)
  • bioreactors (large-scale production)

2
Secondary metabolites from plant cell cultures
  • Features of these compounds
  • low molecular wt
  • synthesis (in plant cell) is tightly regulated
  • A few products are commercially produced
  • examples shikonin, ginseng compounds, berberine
  • more are not produced because of two main
    problems
  • low yields in in vitro culture
  • feedback inhibition from SMs stored
    intracellularly

3
Secondary metabolites from plant cell cultures
  • Transport mechanisms in cultivated plant cells
  • cells that transport SMs to vacuolar space for
    storage
  • final yield limited to storage capacity of medium
  • cells that secrete SMs into the medium
  • final yield limited to properties of the medium
  • production is more easily manipulated, but
    separation of culture medium from cultures is
    required
  • SMs originating from cells in the medium,
    secreted into the medium, taken up by other cells
    and stored

4
Secondary metabolites from plant cell cultures
  • Techniques for increasing yields
  • usually attempt to redirect transport
  • permeabilization
  • objective is to permeabilize tonoplast and/or
    cell membrane to drive secretion of SM into the
    culture medium
  • chemical agents such as DMSO or Triton X-100
  • problem poor viability after treatment
  • two-phase systems

5
Secondary metabolites from plant cell cultures
  • Techniques for increasing yields
  • two-phase systems
  • strategy to provide an artificial site for SM
    accumulation
  • example sanguinarine from cultures of
    California poppy cells
  • sanguinarine is an alkaloid that acts as an agent
    against dental caries

6
Secondary metabolites from plant cell cultures
  • example sanguinarine from California poppy
    cells
  • sanguinarine is an alkaloid that acts as an agent
    against dental caries
  • cells are grown in a 2-liter air-lift bioreactor
  • this bioreactor uses 2 phases an aqueous culture
    phase and a silicone oil extraction phase
  • bioreactor is sparged thru a central draft tube
    gas bubbles are disengaged thru a top layer of
    silicone fluid

7
Secondary metabolites from plant cell cultures
  • example sanguinarine from California poppy
    cells
  • difference in specific gravity between aqueous
    and extraction phases, as well as gentle mixing
    in the airlift system maintains a clean
    separation between phases
  • a small contact area between phases is sufficient
    for secretion into the extraction phase
  • advantages (of this two-phase system)
  • silicone extraction phase doesn't affect cell
    viability

8
Secondary metabolites from plant cell cultures
  • example sanguinarine from California poppy
    cells
  • advantages (of this two-phase system)
  • SM product is isolated rapidly
  • SM product can be localized in a reduced volume
  • elicitation can be incorporated
  • biosynthetic enzymes induced (usu. with an
    oligosaccharide)
  • increase in SM production results

9
Secondary metabolites from plant cell cultures
  • Other systems
  • immobilized cell culture (2 approaches)
  • entrapment in alginate beads
  • adherence of cells to a porous membrane
  • in both cases, the objectives
  • control of growth (high nutrient levels first,
    then elicitation or starvation)
  • release of SM into the medium where it can be
    harvested

10
Secondary metabolites from plant cell cultures
  • Other systems
  • hairy root culture culture of fast-growing
    roots by
  • inoculation with Agrobacterium rhizogenes (esp.
    useful where differentiated cells are required
    for SM production)
  • transfer of T-DNA causing hairy roots, then
    elimination of Agrobacterium by antibiotic
  • (transformed) hairy roots are first subcultured
    to solid, then liquid medium

11
Secondary metabolites from plant cell cultures
  • Other systems
  • hairy root culture (contin)
  • SM is released into the medium, then harvested
  • primary advantage a simple system w/o problems
    inherent with cell suspensions
  • limited to spp. susceptible to A. rhizogenes
  • biotransformation

12
Secondary metabolites from plant cell cultures
  • Other systems
  • biotransformation
  • chemical conversion of an exogenously supplied
    substance by living cell cultures
  • example addition of hydroquinone (inexpensive
    precursor) into liquid culture medium containing
    vinca cells results in efficient production of
    arbutin (a skin depigmentation agent)

13
Secondary metabolites from plant cell cultures
  • Outlook for SM production in plant cell cultures
    (where plant cell systems may be preferred over
    whole plants)
  • rare and endangered plants of pharmaceutical
    interest
  • plants that grow too slowly to match a sudden
    demand (e.g., Taxol present only in the bark of
    Taxus brefolia)
  • some cell cultures can be made to produce a
    single compound, e.g., sanguinarine from poppy
    cell culture
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