Transcript Document 7210353

Designing an organism that uses
light energy for the production of
EtOH
Antón Vila-Sanjurjo & Carlos Bustamante
Intermembrane Space
Mitochondrial
Matrix
• ATP synthase captures the chemical energy
released by the burning of biological
molecules.
• Would it be possible to run the ATP
synthase in response to light in
mitochondria?
Intermembrane Space
Mitochondrial
Matrix
Our proposal
-Insert the proton pump bacteriorhodopsin
in the yeast inner mitochondrial membrane so that
it creates a proton gradient in response to light.
-This gradient could then be used by the
ATP synthase to make ATP.
-Use the ATP to fix CO2 from the air.
-Direct this fixed CO2 to the production of
EtOH.
Kuhlbrandt W.
Nature. 2000 Aug 10;406(6796):569-70.
-It consists of seven membranespanning helical structures.
-contains one molecule of a linear
pigment called retinal, one end of which
is attached to a lysine residue in helix
G.
a, Light-induced isomerization of the
protonated retinal from all- trans
(purple) to 13-cis (pink) triggers the
transfer of the proton to aspartate 85,
aided by a slight movement of this
residue in the L intermediate (b)
towards the nitrogen atom. In the M
state (c), the deprotonated retinal
(yellow) straightens, pushing against
helix F and causing it to tilt. This opens
a channel on the inner, cytoplasmic side
of the membrane through which
aspartate 96 is reprotonated (d), having
given up its proton to the nitrogen on
the retinal. Aspartate 85 transfers its
proton through a network of hydrogen
bonds and water molecules to the
outside medium, past arginine 82, which
has moved slightly
Eur. J. Biochem. 218, 377-383 (1993)
Purification of ATP synthase from beef heart mitochondria (F,F,) and co-reconstitution with monomeric
bacteriorhodopsin into liposomes capable of light-driven ATP synthesis
Barbara DEISINGER, Thomas NAWROTH, Klaus ZWICKER, Simone MATUSCHKA, Gabriele JOHN,
Guido ZIMMER and Hans-Joachim FREISLEBEN
“…The illumination of those
Liposomes with narrow-band filtered
light of high intensity resulted in an
energization of the liposomes. As
shown earlier with yeast and
bacterial
enzymes under similar conditions,
native ATP synthases show lightdriven
ATP synthesis for a long period of
time,
e.g. 30 min…”
All-trans Retinal cofactor
Biosynthetic
pathway to retinal
Ronald F. Peck, Eric A. Johnson, and Mark P. Krebs
JOURNAL OF BACTERIOLOGY, June 2002,
p. 2889–2897 Vol. 184, No. 11
Biosynthetic
pathway to retinal
Ronald F. Peck, Eric A. Johnson, and Mark P. Krebs
JOURNAL OF BACTERIOLOGY, June 2002,
p. 2889–2897 Vol. 184, No. 11
-Yeast cannot make any of
these compounds!!!
Synthesis of Carotenoids
in Erwinia species
Norihiko Misawa* and Hiroshi Shimada
Journal of Biotechnology, Volume 59, Issue 3 , 3 January 1998, Pages 169181
Synthesis of Carotenoids
in Erwinia species
Norihiko Misawa* and Hiroshi Shimada
Journal of Biotechnology, Volume 59, Issue 3 , 3 January 1998, Pages 169181
-The food yeast S. cerevisiae which is
not able to synthesize carotenoids, is
known to accumulate ergosterol as its
principal isoprenoid compound.
-The biosynthetic pathway specific to
ergosterol branches at FPP (farnesyl
pyrophosphate)
-Thus, it may be feasible to direct the
carbon flux for the ergosterol
biosynthesis partially to the pathway for
carotenoid production by the
introduction of the carotenogenic genes
starting with the Erwinia crtE gene.
Synthesis of Carotenoids
in Saccharomyces
cerevisiae
Norihiko Misawa* and Hiroshi Shimada
Journal of Biotechnology, Volume 59, Issue 3 , 3 January 1998, Pages 169181
-Plasmid Y5143 was constructed by
inserting the E. uredovora crtE, crtB, crtI,
and crtY genes, which were flanked by the
promoters and terminators derived from the
S. cerevisiae PGK (phosphoglycerate
kinase), GAL7, and GAP (glyceraldehyde-3phosphate dehydrogenase)
-The S. cerevisiae R7 transformant
harboring Y5143 accumulated 103 mg g1
dry weight of beta-carotene along with small
amounts of the intermediary carotenoid
metabolites in the stationary phase
Biosynthetic
pathway to retinal
Ronald F. Peck, Eric A. Johnson, and Mark P. Krebs
JOURNAL OF BACTERIOLOGY, June 2002,
p. 2889–2897 Vol. 184, No. 11
-The deletion of a single gene (brp)
simultaneously results in decreased
retinal
accumulation and increased betacarotene
accumulation.
-When brp and blh are both deleted,
betacarotene accumulation increases further
and
no retinal is detectable.
-Thus, b-carotene is likely to be the
precursor to retinal in H. salinarum and
is
not converted spontaneously to retinal.
-Brp and Blh appear to have redundant
functions.
-The redundancy may be needed to
allow
Brp/blh
What do we want this ATP for?
• We could use it to fix CO2.
What do we want this ATP for?
• We could use it to fix CO2.
• The CO2 could then be used for the
production of EtOH.
What do we want this ATP for?
• We could use it to fix CO2.
• The CO2 could then be used for the
production of EtOH.
• How do we engineer yeast so they can fix
CO2?
Krebs Cycle
The
Krebs
cycle
reactions
produce
CO2
TCA Cycle during
Alcoholic
Fermentation in yeast
-Although the TCA cycle is mainly
repressed during fermentation, there is
residual TCA activity to fuel
biosynthetic reactions.
-The cycle operates in 2 branches:
-Reductive, leading to
fumarate formation.
-Oxidative, leading to 2-OG
formation.
Carole Camarasa, Jean-Philippe Grivet and Sylvie Dequin
Microbiology (2003), 149, 2669–2678
Pyruvate
carboxylase
TCA Cycle during
Alcoholic
Fermentation in yeast
FIG. 1. Biochemical reaction network for
yeast central carbon metabolism. The arrows
indicate reaction directionality. Letters in
boldface type indicate metabolites for which
the 13C-labeling pattern can be accessed
through METAFoR analysis. Abbreviations:
G6P, glucose-6-phosphate; F6P, fructose-6
phosphate; P5P, pentose-5-phosphates; E4P,
erythrose-4 phosphate; S7P, seduheptulose-7
phosphate; G3P, glyceraldehyde-3-phosphate;
PGA, 3-phosphoglycerate; ICT, isocitrate;
OGA, oxoglutarate; SUC, succinate; MAL,
malate; GOX, glyoxylate.
Jocelyne Fiaux,1 Z. Petek Çakar,2, Marco
Sonderegger,2 Kurt Wüthrich,1 Thomas Szyperski,3*
and Uwe Sauer2
Eukaryotic Cell, February 2003, p. 170-180,
Vol. 2, No. 1
TCA Cycle during
Alcoholic
Fermentation in yeast
Jocelyne Fiaux, Z. Petek Çakar, Marco
Sonderegger, Kurt Wüthrich, Thomas
Szyperski, and Uwe Sauer
Eukaryotic Cell, February 2003, p. 170-180,
Vol. 2, No. 1
TCA Cycle during Alcoholic Fermentation in
yeast
•
•
Metabolic Flux Variation of Saccharomyces cerevisiae Cultivated in
a Multistage Continuous Stirred Tank Reactor Fermentation
Environment
Yen-Han Lin, Dennis Bayrock, and W. Michael Ingledew
1055 Biotechnol. Prog. 2001, 17, 1055-1060
“… the anaplerotic pathway that transports cytosolic OAA
across the mitochondrial membrane to become mitochondrial
OAA (the fluxes funneling through this pathway
were 14.8%, 16.19%, 9.26%, and 8.23% from F1 to F4,
respectively) is far more active than its counterpart
pathway regulated by PDH* (fluxes of 2.82%, 3.09%,
1.78%, and 1.59%) connected to the TCA cycle. This
indicated that the TCA cycle was mainly replenished
through the reaction catalyzed by PYC…”
*pyruvate dehydrogenase (PDH;converting pyruvate to acetyl-CoA and CO2. Point of entry of
carbon into the oxidative, regular TCA cycle).
• Bacteriorhodopsin can drive ATP synthesis
in vitro.
• Yeast has reactions that can fix CO2 and
these reactions are active during alcohol
fermentation.
• Any more evidence????
Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the
inner mitochondrial membrane of Schizosaccharomyces pombe
Hoffmann,A.; Hildebrandt,V.; Heberle,J.;Buldt,G.
Proc.Natl.Acad.Sci.U.S.A.,1994, 91, 20, 9367-9371
Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the
inner mitochondrial membrane of Schizosaccharomyces pombe
Hoffmann,A.; Hildebrandt,V.; Heberle,J.;Buldt,G.
Proc.Natl.Acad.Sci.U.S.A.,1994, 91, 20, 9367-9371
“…Transformed yeast cells (clone pEPVp-COXIV) were grown under anaerobic conditions with
and without light. Glucose was given to the
culture medium as energy source... The ratio of
glucose concentrations in the culture media of
cells grown with and without illumination is
plotted in Fig. 5 for clone pEPVp-COX-IV
(dotted line)... About 20h after inoculation, when
fermentation of this clone starts, the
concentration of glucose in the culture medium
of these cells, grown with light, increased in
comparison to the corresponding culture kept in
the dark... The observed effect can be interpreted
by the production of ATP due to the light-induced
proton gradient across de IM. Therefore, less ATP
resulting from anaerobical glucose consumption
is needed...“
Michael Hügler, Harald Huber, Karl Otto Stetter,
and Georg Fuchs
Autotrophic CO2 fixation pathways in archaea
(Crenarchaeota)
Arch Microbiol (2003) 179 : 160–173
Fig. 1A–D Outlines of the four
known pathways for autotrophic
CO2 fixation. The reactions
catalyzed by key enzymes of
these pathways are indicated by
bold arrows.
A Calvin-Bassham-Benson cycle;
B reductive citric acid cycle
rTCA;
C reductive acetyl-CoA pathway;
D 3-hydroxypropionate cycle.
-One complete turn of the rTCA cycle
yields one molecule of oxaloacetate
from four molecules of CO2,
regeneration of the acceptor molecule
of the cycle, oxaloacetate.
-Key enzymes of the reductive citric
acid cycle are 2oxoglutarate:ferredoxin
oxidoreductase and ATP citrate lyase.
rTCA
Our proposal
-Insert the proton pump bacteriorhodopsin
in the yeast inner mitochondrial membrane so that
it creates a proton gradient in response to light.
-This gradient could then be used by the
ATP synthase to make ATP.
-Use the ATP to fix CO2 from the air.
-Direct this fixed CO2 to the production of
EtOH.