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