Transcript Seminar on Oil accumulation in Chlamydomonas

Algal Lipid Bodies: Stress Induction, Purification, and
Biochemical Characterization in Wild-Type and Starchless
Chlamydomonas reinhardtii
Zi T. Wang*, Nico Ullrich, Sunjoo Joo*, Sabine Waffenschmidt, and
Ursula Goodenough* (Washington University*, Institut fur Biochemie,
Universitat zu Koln, Germany)
Eukaryotic Cell, 2009, 8:1856–1868
Ursula
Goodenough
Sabine Waffenschmidt
BIOFUELS (A role for algae?)
• Potential to have no net increase in [CO2] in atmosphere
• Renewable and sustainable
• Ethanol
–
Produced now from corn starch (sugar cane in Brazil) by
fermentation w/yeast or bacteria and distillation
• Biodiesel
–
Produced now from vegetable oil (triacylglycerols or
triglycerides), less polluting than Petrodiesel
• Other possible fuels
–
Butanol
–
Long-chain hydrocarbons
–
Hydrogen (H2) (combustion does not produce any
greenhouse gas)
Soybean
Corn
Sunflower
Safflower
Peanut
Cottonseed
Rapeseed (canola)
Olive
Palm
Coconut
Chemical conversion step: Oil (triglycerides) to
Biodiesel
R= 16-22 carbons
Petro Diesel: C10H20 to C15H28
Chlamydomonas reinhardti: A model system for
Biofuel production
Chlamydomonas reinhardtii
• Model genetic organism for photosynthesis/bioenergetics and cell motility
• Grows rapidly, autotrophically or heterotrophically
• Controlled sexual or asexual reproduction
• All 3 genomes have been sequenced and are transformable:
1. Nuclear (125,000 KB; 15,000 genes)
2. Chloroplast (200 KB; 100 genes)
3. Mitochondrion (16 KB; 12 genes)
• Can knock-down genes with RNAi
FIG. 1. Confocal microscopy surveys of cw15 (A) and cw15 sta6 (B) cell
samples starved for N for 24 h. Red, chlorophyll autofluorescence;
yellow, Nile Red fluorescence
cw15
cw15 sta6
(no starch
synthesis)
Wang, Z. T. et al. 2009. Eukaryotic Cell 8(12):1856-1868
Conclusion: nitrogen starvation increases lipid bodies
(LBs), and so does knocking out starch synthesis
FIG. 2. (A) Optical sections of cw15 cells (top) and cw15 sta6 cells (bottom)
starved for N for 24 h. (B) Three-dimensional reconstructions of through-focal
optical sections of cw15 cells (top) and cw15 sta6 cells (bottom) starved for N
for 24 h. Red, chlorophyll autofluorescence; yellow, Nile Red fluorescence
cw15
sta6
Wang, Z. T. et al. 2009. Eukaryotic Cell 8(12):1856-1868
LBs are in the cytoplasm, sticking to the chloroplast surface
Movies
Figures A1 & A2. Through-focal optical
sections of two cw15sta6 cells N-starved for 48
h. Red, chlorophyll autofluorescence; yellow,
Nile-Red fluorescence.
FIG. 3. Size distributions
of LBs from:
(A) through-focal optical
sections of cw15 cells
starved for N for 24 h
and cw15 sta6 cells
starved for N for 24 and
48 h
Popped cells after 24 h
of N starvation (B),
and washed LBs after 18
h of N starvation (C)
FIG. 4. Confocal fluorescence microscopy images of cw15 sta6 cells
popped in situ
Used this Popped-cell assay to do relative quantification
of LBs.
FIG. 5. Popped
cw15 and cw15
sta6 cells stained
with Nile Red after
24 and 48 h of N
starvation.
Nos. are based on
summed area of
fluorescence
pixels/cell
Wang, Z. T. et al. 2009. Eukaryotic Cell 8(12):1856-1868
FIG. 6. Pooled
distributions of LB
contents in popped
cw15 and cw15
sta6 cells after 0,
24, and 48 h of N
starvation
sta6 data skewed to
the right; authors
suggest LB
production may be
limited by cell
autolysis/autophagy
in the 48 h culture.
“moribund”
FIG. 7. Washed LB
preparation
FIG. 8. (A and B) MS-GC
spectra of Fatty acids
derived from TAG, in
washed LB
preparations from cw15
(A) and cw15 sta6 (B)
cells
Conclusions:
90% of the LB is TAG,
10%
C16-C18 species only,
no longer-chain FA
FIG. 10. Thin-layer chromatographs (TLC) of NGLs and CGLs
from cw15 cells and from initial LB preparations from cw15 sta6
cells
LBs do not have much contamination with plastid lipids
(NGLs), consistent with an ER origin.
FIG. 11. TAG contents of five independent washed LB preparations from cw15 sta6 and
cw15 cells after 18 h of N starvation
Estimate yield of ~ 400 mg TAG/liter of culture
(107 cells/ml) with cw15 sta6
Strengths and weaknesses of this paper:
Strengths:
1. Quality of data/results is high
2. Novel finding (Chlamy was thought to not be a
good TAG accumulator)
3. Variety of methods used (and developed)
4. It is relevant to Biofuels.
Weaknesses:
1. Have to centrifuge cells and replace the medium
(not practical at large scale).
2. Did not detect proteins clearly assoc. with LBs.
3. Use of vague term (“Moribund”) cells to explain LB
size distribution in sta6 after 48 h.