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BacteriaBacteria
• Single cellsSingle cells• Small size (1-5 Small size (1-5 m)m)• Rapid reproductionRapid reproduction• Genomic and genetic Genomic and genetic
capabilitiescapabilities
Bacterial DiversityBacterial Diversity
• 4 billion years of 4 billion years of evolutionevolution
• Ability to thrive in Ability to thrive in extreme extreme environmentsenvironments
• Use nutrients Use nutrients unavailable to other unavailable to other organismsorganisms
• Tremendous catalytic Tremendous catalytic potentialpotential
Problem to be Solved: Waste Problem to be Solved: Waste Minimization in the Chemical IndustryMinimization in the Chemical Industry
•Most of our manufactured goods involve chemicals
•Chemical industry currently based on chemicals derived from petroleum
Not renewable resourceMany produce hazardous wastes
Use bacteria as the factories of the future
Bacteria as FactoriesBacteria as Factories
Starting materials
Harnessing Catalytic Potential of Harnessing Catalytic Potential of BacteriaBacteria
• Use bacteria as self-replicating multistage Use bacteria as self-replicating multistage catalysts for chemical productioncatalysts for chemical production
• Environmentally benignEnvironmentally benign• Renewable starting materials (feedstocks)Renewable starting materials (feedstocks)
Starting materialsValue-added products
Potential FeedstocksPotential Feedstocks
Characteristics: Characteristics: InexpensiveInexpensive
AbundantAbundant
RenewableRenewable CandidatesCandidates SourceSource• Glucose CGlucose C66HH1212OO66 agricultural wastesagricultural wastes
• Methane CHMethane CH44 natural gas, sewagenatural gas, sewage
• Methanol CHMethanol CH33OHOH methanemethane
• Carbon dioxide/water COCarbon dioxide/water CO22/H/H22O O
atmosphere/photosynthesisatmosphere/photosynthesis
Potential ProductsPotential Products
• FuelsFuels• HH2 2 hydrogen hydrogen
• CHCH44 methane methane
• CHCH33CHCH22OH ethanolOH ethanol
Potential ProductsPotential Products
• Natural products (complex synthesis)Natural products (complex synthesis)• VitaminsVitamins • Therapeutic agentsTherapeutic agents • Pigments Pigments • Amino acidsAmino acids• ViscosifiersViscosifiers• Industrial enzymesIndustrial enzymes• PHAs (biodegradable plastics)PHAs (biodegradable plastics)
Potential ProductsPotential Products
• Engineered productsEngineered products• Starting materials for polymers (such as Starting materials for polymers (such as
rubber, plastics, fabrics)rubber, plastics, fabrics)• Specialty chemicals (chiral)Specialty chemicals (chiral)• Bulk chemicals (CBulk chemicals (C44 acids) acids)
Problem to SolveProblem to Solve
• If bacteria are such wonderful alternatives, why If bacteria are such wonderful alternatives, why are our chemicals still made from are our chemicals still made from environmentally hazardous feedstocks?environmentally hazardous feedstocks?
Bacterial processes are too expensiveBacterial processes are too expensive
Nature’s Design SolutionsNature’s Design Solutions
• Competitive advantage in natural nichesCompetitive advantage in natural niches
• Optimized parametersOptimized parameters• Low nutrientsLow nutrients• Defense systemsDefense systems
OpportunityOpportunity
Redesign bacteria with industrially-valuable Redesign bacteria with industrially-valuable parameters optimizedparameters optimized
• Redirect metabolism to Redirect metabolism to
specific productsspecific products• Increase metabolic efficiencyIncrease metabolic efficiency• Increase process efficiencyIncrease process efficiency
This idea has been around for 30 years, why has the problem not been solved?
Metabolism as a NetworkMetabolism as a Network
• Metabolism: the Metabolism: the complex network of complex network of chemical reactions in chemical reactions in the cellthe cell
• Must redesign the Must redesign the networknetwork
• Understand the Understand the connections to achieve connections to achieve end resultend result
What’s New?What’s New?• GenomicsGenomics
• Bacterial genomes small (1000 = human)Bacterial genomes small (1000 = human)• Hundreds of bacterial genome sequences Hundreds of bacterial genome sequences
availableavailable• Provides the blueprint for the organism (the parts Provides the blueprint for the organism (the parts
list) list)
New platform for redesignNew platform for redesign
What’s New?What’s New?
• Increased understanding of how new kinds Increased understanding of how new kinds of metabolism aroseof metabolism arose
New strategies for redesignNew strategies for redesign
How Build Novel Metabolic How Build Novel Metabolic Pathways?Pathways?
• Whole metabolic pathways: no single gene or Whole metabolic pathways: no single gene or small number of genes confer selective small number of genes confer selective advantageadvantage
• Cannot build a step at a timeCannot build a step at a time
Dilemma: how were entire pathways constructed Dilemma: how were entire pathways constructed during evolution?during evolution?
Modular Aspect of MetabolismModular Aspect of Metabolism
• Metabolic capabilities were built in blocks, Metabolic capabilities were built in blocks, like puzzle pieceslike puzzle pieces
Strategy: Understand the modules and their connectionsRedesign in blocks
Methanol as an Alternative Methanol as an Alternative BiofeedstockBiofeedstock
• Soluble in waterSoluble in water
• InexpensiveInexpensive CHCH33OHOH
• Pure substratePure substrate
• Bacteria that use it Bacteria that use it chemicalschemicals
well-studiedwell-studied
Methylotrophic BacteriaMethylotrophic Bacteria
CH3OH (methanol)
CO2, H2O, cells
O2
Specified product
ApproachApproach
• Define functional Define functional modules by modules by experimental and experimental and evolutionary analysisevolutionary analysis
methanol
Lcyt cCH OH3
MEDHHCHO
HCHO
amicyaninCH NH3 2
MADH
DissimilationMethylene H4MPT
Methenyl H4MPT
N5-Formyl H4MPT
Formyl MFR
CO2
H4MPT
NADHNADPH
2H
Assimilation
Serine cycle
C3 Compounds
Methylene H4F
H4F
CO2
CO2
N10-Formyl H4F
Methenyl H4F
FormateNADH
ATP
NADPH
PurinesfMet-tRNA
•Optimize process parametersOptimize process parameters
x
•Manipulate modules Manipulate modules to optimize productto optimize product product
CO2
BIOMASS
Methylobacterium extorquens AM1
•Grows on one-carbon compounds (reducing power limited)
•Also grows on multi-carbon compounds (ATP-limited)
•Natural habitat: leaf surfaces
•Substantial toolkit for genetic analyses
•Genome sequence available
•Whole genome microarrays available Clover leaf print showing pink Methylobacterium strains
Target Product: Biodegradable Target Product: Biodegradable PlasticsPlastics
CH3OH
CO2
Energy metabolism (dissimilation)C3
BiomassBiomass
Biosynthesis (assimilation)
PHA (biodegradable plastic)
Methylotrophic Metabolic ModulesMethylotrophic Metabolic Modules
Methanol
Formaldehyde
Methylene H4F
Formate
CO2
Methanol Oxidation
H4F-linked
C1 transfer
H4MPT-linked
C1 transfer
FDH1 FDH2 FDH3
Serinecycle
TCAcycle
PHAcycle
GlyoxylateRegenerationcycle
CELLS
PHA
ConstraintsConstraints
• Understanding how the system is Understanding how the system is integrated in time and spaceintegrated in time and space
• Changing how it worksChanging how it works
Work in ProgressWork in Progress• Use genome-wide Use genome-wide
techniques to assess techniques to assess expression of genes expression of genes within each modulewithin each module
• Use metabolic modeling Use metabolic modeling to make predictions about to make predictions about flow through each module flow through each module
• Use labeling techniques Use labeling techniques to measure flow through to measure flow through each moduleeach module
Results: redesign the metabolic network to overproduce a biodegradable plastic
CO2
BIOMASS
Multi-tiered datasetsMulti-tiered datasets
microarrays: mRNAYoko Okubo, Betsy Skovran
proteomics: proteinsJulia Vorholt groupMurray Hackett group
FluxesChris MarxSteve Van DienGreg Crowther
CH3OH HCHO
HCHO Methylene-H4F
NADPH
ATP
Formate
GlycineSerine
2-PG
Malyl-CoA
Glyoxylate
α-KG Succ-CoA
Succinate
Malate
3-PG
Triose-P
6F P
6G P 5R P
4E P
2 NADPH
Cell membrane
CO2 NADH
Hydroxybutyryl-CoA
Ac-CoA
NADPH
PHB
Butyryl-CoA
NADPH
Propionyl-CoA
2 NADH
FADH2
OAA
Pyruvate
Citrate
Ac-CoA
PEP
NADH
Serine Cycle
TCACycle
Acetyl-CoAConversionPathway
PP Pathway
Methylene-H4MPT
CO2NADPH2 e-
CO2NADH2 e-
2e-
NADH 4 H+ext
2 H+ext ATP
: 4.98Biomass yield
10.00
3.84
6.175.55
0.62
0
19.3
3.21
3.17
0.35
0.35
0.30
0.29
0
0.01
0.03
0
0.040.09
0.090
0.21
2.832.56
2.273.27
0
1.00
1.00
1.00
1.00
0.46
2.92
3.27
3.27
0.62
0.56
CO2
CH3OH HCHO
HCHO Methylene-H4F
NADPH
ATPFormate
GlycineSerine
2-PG
Malyl-CoA
Glyoxylate
α-KG Succ-CoA
Succinate
Malate
3-PG
Triose-P
6F P
6G P 5R P
4E P
2 NADPH
Cell membrane
CO2 NADH
Hydroxybutyryl-CoA
Ac-CoA
NADPH
PHB
Butyryl-CoA
NADPH
Propionyl-CoA
2 NADH
FADH2
OAA
Pyruvate
Citrate
Ac-CoA
PEP
NADH
Serine Cycle
TCACycle
Acetyl-CoAConversionPathway
PP Pathway
Methylene-H4MPT
CO2NADPH2 e-
CO2NADH2 e-
2e-
NADH 4 H+ext
2 H+ext ATP
: 4.98Biomass yield
10.00
3.84
6.175.55
0.62
0
19.3
3.21
3.17
0.35
0.35
0.30
0.29
0
0.01
0.03
0
0.040.09
0.090
0.21
2.832.56
2.273.27
0
1.00
1.00
1.00
1.00
0.46
2.92
3.27
3.27
0.62
0.56
CO2
Enzyme activitiesXiaofeng Guo
CO2
FDHs
NADH
MtdAMtdA
CHO-H4FFchFch
FtfLFtfL
CH=H4FNADPH
H4F, ATP
H2O
H2O
HCHO
CH3OH
MDH H2O, 2e-
HCOOH
H4MPT
H4MPT
Fae
CH2=H4MPTMtdA, MtdB
Fhc
MchCH=H4MPT
H2O
NAD(P)H
H2O
H2O
CHO-H4MPT
Methylene H4MPT
spont.H4F
CH2=H4FH2OMethylene
H4FSerine
Metabolite poolsXiaofeng Guo
100 120 140 160 180 200 220 240 2600
40
80
120
160
200
240
280
320
360
400
m/z
AbundanceAverage spectra for serine peak
156
228
114 138174
128184101
220242 256
Global AnalysisGlobal AnalysisGlobal analysis provides indepth information
•Transcription of all detectable genes•Production of all detectable proteins•Measurement of all major fluxes•Measurement of 100s of metabolites
Involves a basic assumption, that all cells are roughly in the same physiological state
Growing body of literature shows this is not correct
Final Phase: Study Metabolism in Final Phase: Study Metabolism in Single CellsSingle Cells
• Metabolic studies in averaged Metabolic studies in averaged populations do not capture the populations do not capture the range of metabolic events or range of metabolic events or heterogeneity in subpopulationsheterogeneity in subpopulations
• Difficult to study multiple metabolic Difficult to study multiple metabolic parameters in single cellsparameters in single cells
Need: new technologies to study living individual cells in real time
Single Cell ChallengesSingle Cell Challenges
• Volume of a bacterial cell ~ fl (10Volume of a bacterial cell ~ fl (10-15-15))• Number of DNA molecules ~2-3Number of DNA molecules ~2-3• Number of mRNA molecules for a specific Number of mRNA molecules for a specific
gene ~10-10,000gene ~10-10,000• Total protein amount ~amoles (10Total protein amount ~amoles (10-18-18))• Total moles of specific metabolites ~ amoles Total moles of specific metabolites ~ amoles
(10(10-18-18))• Respiration rates ~fmol/min/cell (10Respiration rates ~fmol/min/cell (10-15-15 ) )
New Interdisciplinary New Interdisciplinary ApproachesApproaches
• Combine Combine • GenomicsGenomics• Computational biologyComputational biology• MEMS (microelectromechanical systems)MEMS (microelectromechanical systems)• Systems integrationSystems integration• NanotechnologyNanotechnology
Microscale Life Sciences CenterMicroscale Life Sciences CenterUniversity of WashingtonUniversity of Washington
• Center of Excellence of Genomic Sciences funded by NIH Center of Excellence of Genomic Sciences funded by NIH NHGRINHGRI
• Co-directed by Mary Lidstrom and Deirdre Meldrum (EE)Co-directed by Mary Lidstrom and Deirdre Meldrum (EE)
• Started August 2001
• Goal:
Study complex processes in individual living cells
Chemists, biologists, engineers working together
•Move, trap, image single cells (9 cell sets x 11)•Control environment, make additions•Measure 4 fluorescent protein fusions•Single-cell proteomics
•Measure substrate-dependent O2 uptake (phosphorescence sensor)
Microscope-based imaging and detectionof fluorescence, phosphorescence
Environmental control
Multi-parameter high throughput analysis at the single-cell level, leading to understanding of metabolic networks
N. Dovichi group (Chemistry); L. Burgess group (Chemistry); D. Meldrum group (Elec Engr); A. Jen group (Mat Sci Engr)
Microsystem-Based Devices for Microsystem-Based Devices for Studying Single CellsStudying Single Cells
Evidence for HeterogeneityEvidence for Heterogeneity• Single-cell cell cycle analysis: growthSingle-cell cell cycle analysis: growth
Tim Strovas,
Linda Sauter 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3
# cells
0
2
4
6
8
10
12
Single Cell Division Times
Time, Hr
Single Cell Division Times During MeOH Growth
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63
Time (hrs)
Range:2.5-4.3 hr
SummarySummary
• Breadth of bacterial diversity provides Breadth of bacterial diversity provides opportunityopportunity
• Environmentally benign aspects provide Environmentally benign aspects provide impetusimpetus
• New approaches provide strategiesNew approaches provide strategies• Result: increasing number of microbially-Result: increasing number of microbially-
based products over the next several yearsbased products over the next several years
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