Bacteria Single cellsSingle cells Small size (1-5  m)Small size (1-5  m) Rapid reproductionRapid reproduction Genomic and genetic capabilitiesGenomic and genetic capabilities

Bacterial Diversity 4 billion years of evolution4 billion years of evolution Ability to thrive in extreme environmentsAbility to thrive in extreme environments Use nutrients unavailable to other organismsUse nutrients unavailable to other organisms Tremendous catalytic potentialTremendous catalytic potential

Problem to be Solved: Waste Minimization in the Chemical Industry Most of our manufactured goods involve chemicals Chemical industry currently based on chemicals derived from petroleum Not renewable resource Many produce hazardous wastes Use bacteria as the factories of the future

Bacteria as Factories Starting materials

Harnessing Catalytic Potential of Bacteria Use bacteria as self-replicating multistage catalysts for chemical productionUse bacteria as self-replicating multistage catalysts for chemical production Environmentally benignEnvironmentally benign Renewable starting materials (feedstocks)Renewable starting materials (feedstocks) Starting materials Value-added products

Potential Feedstocks Characteristics: Inexpensive AbundantRenewable Candidates Source Candidates Source Glucose C 6 H 12 O 6 agricultural wastesGlucose C 6 H 12 O 6 agricultural wastes Methane CH 4 natural gas, sewageMethane CH 4 natural gas, sewage Methanol CH 3 OHmethaneMethanol CH 3 OHmethane Carbon dioxide/water CO 2 /H 2 O atmosphere/photosynthesisCarbon dioxide/water CO 2 /H 2 O atmosphere/photosynthesis

Potential Products FuelsFuels H 2 hydrogenH 2 hydrogen CH 4 methaneCH 4 methane CH 3 CH 2 OH ethanolCH 3 CH 2 OH ethanol

Potential Products Natural products (complex synthesis)Natural products (complex synthesis) VitaminsVitamins Therapeutic agentsTherapeutic agents PigmentsPigments Amino acidsAmino acids ViscosifiersViscosifiers Industrial enzymesIndustrial enzymes PHAs (biodegradable plastics)PHAs (biodegradable plastics)

Potential Products Engineered productsEngineered products Starting materials for polymers (such as rubber, plastics, fabrics)Starting materials for polymers (such as rubber, plastics, fabrics) Specialty chemicals (chiral)Specialty chemicals (chiral) Bulk chemicals (C 4 acids)Bulk chemicals (C 4 acids)

Problem to Solve If bacteria are such wonderful alternatives, why are our chemicals still made from environmentally hazardous feedstocks?If bacteria are such wonderful alternatives, why are our chemicals still made from environmentally hazardous feedstocks? Bacterial processes are too expensive

Nature’s Design Solutions Competitive advantage in natural nichesCompetitive advantage in natural niches Optimized parametersOptimized parameters Low nutrientsLow nutrients Defense systemsDefense systems

Opportunity Redesign bacteria with industrially-valuable parameters optimized Redirect metabolism toRedirect metabolism to specific products specific 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 Network Metabolism: the complex network of chemical reactions in the cellMetabolism: the complex network of chemical reactions in the cell Must redesign the networkMust redesign the network Understand the connections to achieve end result Understand the connections to achieve end result

What’s New? GenomicsGenomics Bacterial genomes small (1000 = human)Bacterial genomes small (1000 = human) Hundreds of bacterial genome sequences availableHundreds of bacterial genome sequences available Provides the blueprint for the organism (the parts list)Provides the blueprint for the organism (the parts list) New platform for redesign

What’s New? Increased understanding of how new kinds of metabolism aroseIncreased understanding of how new kinds of metabolism arose New strategies for redesign

How Build Novel Metabolic Pathways? Whole metabolic pathways: no single gene or small number of genes confer selective advantageWhole metabolic pathways: no single gene or small number of genes confer selective advantage Cannot build a step at a timeCannot build a step at a time Dilemma: how were entire pathways constructed during evolution?

Modular Aspect of Metabolism Metabolic capabilities were built in blocks, like puzzle piecesMetabolic capabilities were built in blocks, like puzzle pieces Strategy: Understand the modules and their connections Redesign in blocks

Methanol as an Alternative Biofeedstock Soluble in waterSoluble in water InexpensiveCH 3 OHInexpensiveCH 3 OH Pure substratePure substrate Bacteria that use it chemicalsBacteria that use it chemicals well-studied well-studied

Methylotrophic Bacteria CH 3 OH (methanol) CO 2, H 2 O, cells O2O2 Specified product

Approach Define functional modules by experimental and evolutionary analysisDefine functional modules by experimental and evolutionary analysis methanol L cyt c CH OH 3 MEDH HCHO amicyanin CH NH 32 MADH Dissimilation Methylene H 4 MPT Methenyl H 4 MPT N 5 -Formyl H 4 MPT Formyl MFR CO 2 H 4 MPT NADH NADPH 2H Assimilation Serine cycle C 3 Compounds Methylene H 4 F H4FH4F CO 2 N 10 -Formyl H 4 F Methenyl H 4 F Formate NADH ATP NADPH Purines fMet-tRNA Optimize process parametersOptimize process parameters x Manipulate modules to optimize productManipulate modules to optimize product product CO 2 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 Plastics CH 3 OH CO 2 Energy metabolism (dissimilation) C3C3 Biomass Biosynthesis (assimilation) PHA (biodegradable plastic)

Methylotrophic Metabolic Modules Methanol Formaldehyde Methylene H 4 F Formate CO 2 Methanol Oxidation H 4 F-linked C 1 transfer H 4 MPT-linked C 1 transfer FDH1 FDH2FDH3 Serine cycle TCA cycle PHA cycle Glyoxylate Regeneration cycle CELLS PHA

Constraints Understanding how the system is integrated in time and spaceUnderstanding how the system is integrated in time and space Changing how it worksChanging how it works

Work in Progress Use genome-wide techniques to assess expression of genes within each moduleUse genome-wide techniques to assess expression of genes within each module Use metabolic modeling to make predictions about flow through each moduleUse metabolic modeling to make predictions about flow through each module Use labeling techniques to measure flow through each moduleUse labeling techniques to measure flow through each module Results: redesign the metabolic network to overproduce a biodegradable plastic CO 2 BIOMASS

Multi-tiered datasets microarrays: mRNA Yoko Okubo, Betsy Skovran proteomics: proteins Julia Vorholt group Murray Hackett group Fluxes Chris Marx Steve Van Dien Greg Crowther Enzyme activities Xiaofeng Guo CO 2 FDHs NADH MtdA CHO-H 4 F Fch FtfL CH=H 4 F NADPH H 4 F, ATP H2OH2O H2OH2O HCHO CH 3 OH MDH H 2 O, 2e - HCOOH H 4 MPT Fae CH 2 =H 4 MPT MtdA, MtdB Fhc Mch CH=H 4 MPT H2OH2O NAD(P)H H2OH2O H2OH2O CHO-H 4 MPT Methylene H 4 MPT spont. H4FH4F CH 2 =H 4 F H2OH2O Methylene H 4 F Serine Metabolite pools Xiaofeng Guo

Global Analysis Global 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 Single Cells Metabolic studies in averaged populations do not capture the range of metabolic events or heterogeneity in subpopulationsMetabolic studies in averaged populations do not capture the range of metabolic events or heterogeneity in subpopulations Difficult to study multiple metabolic parameters in single cellsDifficult to study multiple metabolic parameters in single cells Need: new technologies to study living individual cells in real time

Single Cell Challenges Volume of a bacterial cell ~ fl ( )Volume of a bacterial cell ~ fl ( ) Number of DNA molecules ~2-3Number of DNA molecules ~2-3 Number of mRNA molecules for a specific gene ~10-10,000Number of mRNA molecules for a specific gene ~10-10,000 Total protein amount ~amoles ( )Total protein amount ~amoles ( ) Total moles of specific metabolites ~ amoles ( )Total moles of specific metabolites ~ amoles ( ) Respiration rates ~fmol/min/cell ( )Respiration rates ~fmol/min/cell ( )

New Interdisciplinary Approaches CombineCombine GenomicsGenomics Computational biologyComputational biology MEMS (microelectromechanical systems)MEMS (microelectromechanical systems) Systems integrationSystems integration NanotechnologyNanotechnology

Microscale Life Sciences Center University of Washington Center of Excellence of Genomic Sciences funded by NIH NHGRICenter of Excellence of Genomic Sciences funded by NIH NHGRI 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 O 2 uptake (phosphorescence sensor) 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 Studying Single Cells

Evidence for Heterogeneity Single-cell cell cycle analysis: growthSingle-cell cell cycle analysis: growth Tim Strovas, Linda Sauter Range: hr

Summary Breadth of bacterial diversity provides opportunityBreadth of bacterial diversity provides opportunity Environmentally benign aspects provide impetusEnvironmentally benign aspects provide impetus New approaches provide strategiesNew approaches provide strategies Result: increasing number of microbially- based products over the next several yearsResult: increasing number of microbially- based products over the next several years