Designing%20an%20organism%20that%20uses%20light%20energy%20for%20the%20production%20of%20EtOH - PowerPoint PPT Presentation

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Title: Designing%20an%20organism%20that%20uses%20light%20energy%20for%20the%20production%20of%20EtOH


1
Designing an organism that uses light energy for
the production of EtOH
Antón Vila-Sanjurjo Carlos Bustamante
2
Intermembrane Space
Mitochondrial Matrix
3
  • 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?

4
Intermembrane Space
Mitochondrial Matrix
5
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.

6
  • Kuhlbrandt W.
  • Nature. 2000 Aug 10406(6796)569-70.

-It consists of seven membrane-spanning 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
7
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 synthesisBarbara 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 light-driven
  • ATP synthesis for a long period of time,
  • e.g. 30 min

8
All-trans Retinal cofactor
9
Biosynthetic pathway to retinal
  • Ronald F. Peck, Eric A. Johnson, and Mark P.
    Krebs
  • JOURNAL OF BACTERIOLOGY, June 2002,
  • p. 28892897 Vol. 184, No. 11

10
Biosynthetic pathway to retinal
  • Ronald F. Peck, Eric A. Johnson, and Mark P.
    Krebs
  • JOURNAL OF BACTERIOLOGY, June 2002,
  • p. 28892897 Vol. 184, No. 11
  • -Yeast cannot make any of these compounds!!!

11
Synthesis of Carotenoids in Erwinia
speciesNorihiko Misawa and Hiroshi Shimada
Journal of Biotechnology, Volume 59, Issue 3 , 3
January 1998, Pages 169-181

12
Synthesis of Carotenoids in Erwinia
speciesNorihiko Misawa and Hiroshi Shimada
Journal of Biotechnology, Volume 59, Issue 3 , 3
January 1998, Pages 169-181
-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.
13
Synthesis of Carotenoids in Saccharomyces
cerevisiaeNorihiko Misawa and Hiroshi Shimada
Journal of Biotechnology, Volume 59, Issue 3 , 3
January 1998, Pages 169-181
  • -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-3-phosphate 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

14
Biosynthetic pathway to retinal
  • Ronald F. Peck, Eric A. Johnson, and Mark P.
    Krebs
  • JOURNAL OF BACTERIOLOGY, June 2002,
  • p. 28892897 Vol. 184, No. 11
  • -The deletion of a single gene (brp)
  • simultaneously results in decreased retinal
  • accumulation and increased beta-carotene
  • accumulation.
  • -When brp and blh are both deleted, beta-
  • carotene 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
  • retinal production under both aerobic and
  • anaerobic growth conditions.

Brp/blh
15
What do we want this ATP for?
  • We could use it to fix CO2.

16
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.

17
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?

18
Krebs Cycle
The Krebs cycle reactions produce CO2
19
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, 26692678

20
Pyruvate carboxylase
21
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

22

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

23
TCA Cycle during Alcoholic Fermentation in yeast
  • Metabolic Flux Variation of Saccharomyces
    cerevisiae Cultivated ina Multistage Continuous
    Stirred Tank Reactor FermentationEnvironmentYen-
    Han Lin, Dennis Bayrock, and W. Michael
    Ingledew1055 Biotechnol. Prog. 2001, 17,
    1055-1060
  • the anaplerotic pathway that transports
    cytosolic OAAacross the mitochondrial membrane
    to become mitochondrialOAA (the fluxes funneling
    through this pathwaywere 14.8, 16.19, 9.26,
    and 8.23 from F1 to F4,respectively) is far
    more active than its counterpartpathway
    regulated by PDH (fluxes of 2.82, 3.09,1.78,
    and 1.59) connected to the TCA cycle.
    Thisindicated that the TCA cycle was mainly
    replenishedthrough the reaction catalyzed by
    PYC
  • pyruvate dehydrogenase (PDHconverting pyruvate
    to acetyl-CoA and CO2. Point of entry of carbon
    into the oxidative, regular TCA cycle).

24
  • 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????

25

26

27
Photoactive mitochondria in vivo transfer of a
light-driven proton pump into the inner
mitochondrial membrane of Schizosaccharomyces
pombeHoffmann,A. Hildebrandt,V.
Heberle,J.Buldt,G.Proc.Natl.Acad.Sci.U.S.A.,1994
, 91, 20, 9367-9371
28
Photoactive mitochondria in vivo transfer of a
light-driven proton pump into the inner
mitochondrial membrane of Schizosaccharomyces
pombeHoffmann,A. Hildebrandt,V.
Heberle,J.Buldt,G.Proc.Natl.Acad.Sci.U.S.A.,1994
, 91, 20, 9367-9371
  • Transformed yeast cells (clone pEPVp-COX-IV)
    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...

29
  • Michael Hügler, Harald Huber, Karl Otto Stetter,
    and Georg Fuchs
  • Autotrophic CO2 fixation pathways in archaea
    (Crenarchaeota)
  • Arch Microbiol (2003) 179 160173
  • Fig. 1AD 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 2-oxoglutarateferredoxin oxidoreductase and
    ATP citrate lyase.

rTCA
30
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.
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