synthetic biology and light-dependent systems - arpa-e...the synthetic biology platform microbes...
TRANSCRIPT
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Synthetic Biology and Light-Dependent
SystemsPamela Silver
Dept of Systems BiologyHarvard Medical School
Wyss Institute of Biologically Inspired Engineering
Director, Harvard University Graduate Program in Systems Biology
http://silver.med.harvard.edu/
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The synthetic biology platform
Microbes that process sunlight and CO2
Photosynthetic ecosystems and organisms
Designing Biological Systems for Programed Interface with the Environment
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Can we make Biology easier to engineer?
Rapid, inexpensive DNA synthesis
Sequenced genomes for raw materials
Information explosion via the internet
Worldview from computer chip designâ Appreciation of biological modularity
eg promoters, genes, proteins
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Biological Modularity
Examples of modularity:
Genes (promoters, ORFs, introns, enhancers)
RNA (Translation, stability, export, localization)
Proteins (Targeting, DNA binding, dimerization, degradation) Pathways (Signaling, metabolism)
Biological design can test the limits of modularity
What does Nature have to offer?
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Bacterial Devices - Abstraction
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partsregistry.org
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igem.org
Example Part
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Can we make biological design predictable?
⢠Standardized parts
⢠Measurements of behavior
⢠Mathematical models
⢠Gene to genome synthesis - when does the âexperimentâ start?
- Consider the future synthetic biologistâŚâŚ..
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Designing Light-Dependent BioSystems
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Engineering microorganisms for energy production
Conclusions from the JASON report:
Boosting efficiency of fuel formation form microorganisms is THE major technological application of Synthetic Biology
Engineering fuel production from microbes is a SYSTEMS problem Successful engineering requires a basic understanding of the system to be engineered (multiple feedback loops, etc)
Study Leader Mike Brenner; 6/23/06
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Photosynthetic organisms come in all shapes and sizes
alga
non-vascular plantC4 plant
102 m
cyanobacterium
10-6 m
Cyanobacteria are responsible for ~50% of all photosynthesis on earth!
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Reaction
glyceraldehyde 3-phosphate
carbon-fixation cycle
Ribulose-1,5-bisphosphate
CO2
ATP
ADP
NADPH
NADP
carbohydrate, fatty acid, amino acid metabolism
light reactions
3-phosphoglycerate
rubiscorubisco
CO2 / O2
3-phosphoglycerate / phosphglycolate h!
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spatial organization is important for engineering and biology
AT ACP KSAT KR
ACP KSAT KR
ACP
SO
SO
HO
SO
HO
HO
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carboxysomes
cell membrane
thylakoid
gas vesicles
polyphosphate granule
Photosystems
Cyanobacteria contain cytoplasmic structures
Stanier et al. 1977
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Carboxysomes are a large organized unit
Concentrate Rubisco+ Carbonic Anhydrase+ CO2 Solution to rubiscoĘźs inefficiency! Resemble viral capsid
Figure from http://upload.wikimedia.org/wikipedia/commons/5/54/Carboxysome_3_images.png
80-120nm
The enzyme carbonic anhydrase, which catalyzes the production of CO2 from bicarbonate
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The carboxysome is part of a carbon concentrating mechanism
CO2 HCO3-
HCO3-
Carboxysome
This system increases the cellâs affinity for CO2 1000x!
ribulose 1,5 - bisphosphate (5 carbon sugar)
RuBPHCO3-
CO2carbonic
anhydrase
RuBisCO
2 x phosphoglycerate (3 carbon sugar)
Dave S.
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Carboxysomes can be labeled with fluorescent proteins
CcmK
RuBisCox 4500 copies
x 60
copies
x 250 copies
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We can track carboxysomes and cells over many days
20080920
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Even spatial distribution can optimize metabolism
⢠A diffusion-limited reaction will be susceptible to spatial arrangements
Tota
l ene
rgy
prod
uctio
n
Pairwise distance
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Harnessing Bacterial Photosynthesis
~ 11% efficient
½ O2
H2O
2 e-2 e-
ATP NADH Glucose
CO2 Ethanol
2 H+
Calvin cycle
Butanol Biodiesel
½ O2
H2O2 H+
2 H+ + 2 e- H2
2 e-2 e-
Engineered bacterium
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FeS clusters: BiologyĘźs Nanowires
e-
⢠A photon lands on Photosystem I
⢠The electron is excited to enter the iron-sulfur clusters
⢠The electron exits by tunneling into ferredoxin
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ĂHydrogenaseA + Hydrogenase
Ferredoxin
H2
HydrogenaseA
GEF
SulfiteReductase
+
Ă
H2
H2
HydrogenaseRescuesGrowthonMinimalMedia
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Grand Challenge: Can we Efficiently Connect Photosynthesis to Hydrogen Production
Photosystem I
Ferredoxin
Hydrogenase
H2
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13
Platform for chemical synthesis
Enormousadvantageforbiomassâindependentchemicalsynthesis
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Kalscheuer et al. 2006
Wax Synthase can synthesize biodiesel in vivo
alcohol
fatty acid
ethyl laurate (biodiesel)
Wax Synthase/Diacylglycerol transferase
(WS/DGAT) from Acetobacter
1. Need fatty acid
2. Need alcohol
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Thioesterase determines fatty acid chain lengthacetyl-CoA
malonyl-CoA
C4 - ACP
C8 - ACP
C10 - ACP
C12 - ACP
C14 - ACP
C16 - ACP E. coli - C16
fatB
thi
oest
eras
e
Umberllulari californica - C12
Cuphea palustris - C18
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Thioesterases are expressed and functional in Synechococcus
Strain 651S
C12 (lauric acid) C14
C16
C18
GC-MS analysis
50
37
20 kDa
C12 C8 thioesterase
- + - +IPTG
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We are testing three alcohol synthesis pathways
photosynthetic cycle
CO2
pyruvate
2-acetolactate
2,3-dihydroxy-isovalerate
2-ketoisovalerate
isobutyraldehyde
isobutanol
alcohol dehydrogenase
2-keto acid decarboxylase
2-keto acid dehydratase
acetolactate synthase
2-keto acid reducto-isomerase
isobutanolethanol
pyruvate
alcohol dehydrogenase
pyruvate decarboxylase
acetaldehyde
ethanol isopentenyl pyrophosphate
isopentenol
prenyl diphosphate hydrolase
isopentenol
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how does metabolism vary with the circadian rhythm?
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200W/m2
2
Platform for chemical synthesis
CO2
Intermediates
Calvin Cycle Foreign
enzymes
Transporters
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Sugar production in cyanobacteria
CO2
StarchOsmotic stress
Calvin Cycle Invertase
Glu + Fru
Glucose facilitated transport (GLF)
Sucrose
⢠Sugars = feedstock for all metabolic engineering⢠Glucose + fructose mixture = âhigh-fructose corn syrupâ⢠Engineering concept:
â Induce sucrose production by osmotic stress (natural process)â Invertase = enzyme that cleaves sucroseâ GLF = facilitated diffusion transporter for glucose, fructose
4
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Expression of glf and invA in Synechococcus to produce glucose and fructose
sugar in the medium:
Cell growth (IPTG + 300 mM NaCl)
Total sugar produced by day 3 = 40% of dry weight of cells
5
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SynechococcusandE.colicoculture
glf+invA 21+1321
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Feeding E. coli with light (on plates)
100 mM NaCl 200 mM NaCl
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How can we get bacteria into animal cells?
1. Inject intozygote
2. Express Invasin and Listriolysin
3. Phagocytosis
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eye
brain
yolk
glf + invA
Making Photosynthetic Fish?
microinject engineered Synechococcus into zebrafish zygote
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Synechococcus is taken up by macrophages
J774 cells + Synechococcus
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25
Mix of commercial potential and scientific feasibility
$0.018$0.019
$0.023
$0.030
Engineering complexity (# of genes + intangibles)
Isoprene
Hydrogen
Value
$/m2/day
1 2 3 4
Sugar/HFCS
Biodiesel
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Technical step Genetic engineering Large-scale production
2-3 years3-5 years (pilot-scale)
Major challenges
Rational basis for engineeringLight distribution in scale-up Business model to move to fuel productionExcessive top-down control
Need basic researchNeed DOE support Production of higher-value compounds first (or subsidies)Fund multiple competing approaches
Prognostication ?
Getting to a bio-solar energy economy: Thoughts and guesses
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Thanks to many people who have contributed to
these ideas
Current people, collaborators and friends:DAVID SAVAGEDevin Burill, Patrick Boyle, Karmella Haynes, Joe Shih, Max Horner Jake Wintermute, Christina Agapakis, Zeev Waks, Bruno Afonso, Gerald Grandl, Henrike Niederholtmeyer, Bernd Wolfstadter, Buz Barstow, Danny Ducat, Noah Taylor, Ron Milo, Sean Megason, Keiji Nishida, Jeff Way, Kim Di Mora, BBF, Tom Knight, Drew Endy, Randy RettbergFunding:NIH, DOE, DOD, HUCE, WIBIE, NSF SynBerc