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