Bioenergetics: Lecture of May 21, 2009 Introduction to bioenergetics. The thermodynamics of biological energy production Kinetic aspects of bioenergetic processes The molecular and cellular organization of bioenergetic systems Photosynthesis structure of the photosynthetic reaction center photosynthetic electron transfer plant photosystems plant photosystems CO 2 fixation photosynthetic efficiency Biofuels Biofuels Ethanol Respiration and ATP synthesis Haber-Bosch process and biological nitrogen fixation
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Bioenergetics: Lecture of May 21, 2009
Introduction to bioenergetics.
The thermodynamics of biological energy production
Kinetic aspects of bioenergetic processes
The molecular and cellular organization of bioenergetic systems
Photosynthesis
structure of the photosynthetic reaction center photosynthetic electron transfer plant photosystems
Z-scheme for non-cyclic photosynthesis electron transfer
photosystem II
(plant photosystems are depicted “upside-down” relative to bacterial RCs…)
photosystem IIZouni et al Nature 409, 739 (2001)
PDB ID 1FE1
Ferreira, Iverson et al. Science 303, 1831 (2004)
Cofactor arrangement in PSII
P680 th ti f hl h ll i i di i h tP680 - the separation of chlorophyll rings in dimer is somewhat greater than in the RC; shows more monomeric character
Light harvesting antenna system in PSII
oxygen evolution
3
oxygen evolution during photosynthesis
steady state
O2 evolving state
dark adapted state
Buchanan, Gruissem, Jones
Biochemistry and Molecular Biology of Plants
Oxygen evolving center:tetra-manganese cluster
Z i t lGuskov et al
Ferreira, Iverson et al.Science 303, 1831 (2004)
Zouni et al Nature 409, 739 (2001)
Nature Structural Biology 16, 334 (2009)
Photosystem II contains several lipid molecules within the structure
Lipids are probably important for turnover of D1 polypeptide, which is recycled
every 30 min in the celly
RC core
photosystem I p yJordan et al. , Nature 411, 909 (2000)
PDB ID: 1JB0
organization of electron transfer centers in PSI
P700 di f hl h llP700 - dimer of chlorophylls
arrangement of antennae
chlorophylls in PS Ic o op y s S
Dutton et al. Adv. Prot. Chem. 63 (2003)
RubiscoRubisco
Reactions Catalyzed by Rubisco
All Rubisco Enzymes are not specific for CO2 over O2
Many RuBisCo-like enzymes sequencesidentified in soil bacteria and archaea: functions are unknownfunctions are unknown
CO2 metabolism - needs to exchange between gas and solution phases:
CO2 (gas) CO2 (aq) + H2O H2CO3 HCO3- + H+
CO2 (gas) CO2 (aq) ∆G˚’ = + 8.4 kJ/moleCO2 (gas) CO2 (aq) ∆G 8.4 kJ/mole
CO2 (aq) + H2O HCO3- + H+ ∆G˚’ = + 4.7 kJ/mole
reversible hydration catalyzed by carbonic anhydrase
M. thermoautotrophicum CAM. thermophila carbonic anhydrase
Biofuels• Engineer cell metabolic pathways to produce
BiofuelsEngineer cell metabolic pathways to produce alcohol: methanol, ethanol, propanol, butanol
• Cells need fuel/energy: best source is glucosegy g• Cellulose can be degraded to glucose by
cellulases
Degradation of Cellulose is the Rate limiting stepRate limiting step
• Faster Cellulases• More stable cellulases (last longer in
incubator, and thus the total yield of degraded cellulase is greater)
• Microorganisms make cellulosomes which have l diff f ll l d diseveral different types of cellulose degrading
enzymes: Progressive degradation
Cellulases are processive enzymesCellulases are processive enzymes
Cl t idi th llClostridium thermocellum
Corn Stover ParticlesPre-treatment: 190C for 15 min
Pre-treatment changes susceptibility of cellulose to enzyme degradation
Utilization of forestland and agricultural land could produce 1.3 billion tons of pdry biomass
This would produce sufficient biofuels to displace 30 percent of domesticto displace 30 percent of domestic petroleum consumption
Biofuels will be a big part of America’s and the world’s energy futureBiofuels will be a big part of America s and the world s energy future.
Biofuels Initiative:•To make cellulosic ethanol cost competitive with gasoline by 2012. •To replace 30 percent of current levels of gasoline consumption with biofuels by 2030 (or 30x30).
•Broad-based environmental benefitsReduced greenhouse gas emissions Reduced petroleum use in fuel production
•Improved national energy securityImproved national energy securityReduced reliance on foreign sources of energy Decreased threat of supply disruptions due to natural disasters, political instability, and price volatility
Metabolic EngineeringMetabolic Engineering
• Maximize the level of NAD availableMaximize the level of NAD available• Cell has to tolerate working concentrations
of substrate and productsof substrate and products
Engineered biochemical pathway to make alcohols from intermediates of the amino acid biosynthesis pathways
Atsumi, Liao Nature (2008) 351: 86-90
Engineered biochemical pathway to make alcohols from intermediates of the amino acid biosynthesis pathways
Atsumi, Liao Nature (2008) 351: 86-90
glucose 2 pyruvate Net reaction:
Engineering Alcohol Dehydrogenase Cofactor Specificity for Isobutanol Production
glucose 2 pyruvate
acetolactate
alsS2 NADH
CO2
Net reaction:
1 glucose + 2 NADPH + 2 NAD+
2 3 dihydroxy isovalerate
acetolactate
ilvCNADPH 1 isobutanol + 2 NADP+ + 2 NADH + 2 CO2
2,3-dihydroxy-isovalerate
keto isovalerate
ilvD
• 66% of glucose carbon results in isobutanol
• 33% carbon lost to CO-keto-isovalerate
isobutyraldehyde
kivDCO2
• 33% carbon lost to CO2
• As constructed the pathway has a co-factor imbalance
EhrlichPathway(Isobutanol)
isobutyraldehyde
i b t l
yqhDNADPH • To produce NADPH the cells have to
spend energy/carbon resulting in lower yield( ) isobutanol yield.
Changing the cofactor specificity of ilvC and yqhD would solve the imbalance.
Metabolic EngineeringMetabolic Engineering
• Maximize the level of NAD availableMaximize the level of NAD available• Cell has tolerate working concentrations of
substrate and productssubstrate and products
Project GoalsProject Goals• Engineer NADH-dependent KARI (ilvC) and IDH (yqhD) enzymes.
D li NADH d d IDH i h 0% f hD NADPH• Deliver a NADH-dependent IDH with 50% of yqhD NADPH-dependent isobutyraldehyde reduction activity by March 2009
• Integrate the engineered enzymes into the isobutanol pathway• Integrate the engineered enzymes into the isobutanol pathway.
• Optimize isobutanol production via strain development.
Clostridium thermocellumClostridium thermocellum– Can completely consume insoluble cellulose
Produces ethanol– Produces ethanol
Produces other products in addition to ethanol– Produces other products in addition to ethanol– Cannot genetically modify Clostridium
Multi-stage biofuel productionMulti stage biofuel production
http://www.ornl.gov/
Evolution of improved endoglucanases
ll l
•Cellulosomal cellulases from anaerobic bacteria Clostridium thermocellum will be parents of recombinant endoglucanases
cellulosomes
thermocellum will be parents of recombinant endoglucanases (EGs)
•Successful optimization of EG and CBH activity enablesSuccessful optimization of EG and CBH activity enables growth on cellulose
Thermostable Cellulases from SCHEMASCHEMA
MTIKEMPQPKTFGELKNL...KETSPIPQPKTFGPLGNL...KQASAIPQPKTYGPLKNL...WRRRGIPGPLGYPLVGSF...WIRKGVKGPRGLPFLGVI...FIRKGIKGPRGFPGIGML...WIRKGVKGPRGFPFFGVIWIRKGVKGPRGFPFFGVI...WIRKGVKGPRGFPFFGVI...WMRKGIKGPRGLPFFGII...WMRKGVKGPRGRPFVGVL...WRRRGVVGPMGFPVLGVF...REKIGLSGPEPHWFLGNL...REKIGLTGPEPHWFMGNL...RSSIGIPGPPVHWLWGNL...KVSKYPKGPLPLPFIGNI E =C KVSKYPKGPLPLPFIGNI...... E =Cijij
300
400
500
600C therophilumH insolensT reeseiHJPlus
Parent 2 Parent 3
Parent 1/u
gE
nzym
e
0
100
200
300ug
Glu
cose
/
50 55 60 65 70 75
Temperature (C)
Directed Evolution of Cyt P450 BM3 to convert propane to propanolconvert propane to propanol
Sugar polymer feedstocksSugar polymer feedstocks
Cellulase synthase can be d i b iexpressed in cyanobacteria
• Cellulase synthaseCellulase synthase can make non-crystalline cellulose