Converting Methanol to Higher Value Added Products with Bacteria by 2mz1O10

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									Bacteria
    •   Single cells
    •   Small size (1-5 mm)
    •   Rapid reproduction
    •   Genomic and genetic
        capabilities
Bacterial Diversity
          • 4 billion years of
            evolution
          • Ability to thrive in
            extreme
            environments
          • Use nutrients
            unavailable to other
            organisms
          • Tremendous 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

                                Value-added products
 Starting materials




• Use bacteria as self-replicating multistage
  catalysts for chemical production
• Environmentally benign
• Renewable starting materials (feedstocks)
          Potential Feedstocks
            Characteristics: Inexpensive
                                   Abundant
                                   Renewable

    Candidates                         Source
•   Glucose C6H12O6                  agricultural wastes
•   Methane CH4                      natural gas, sewage
•   Methanol CH3OH                   methane
•   Carbon dioxide/water CO2/H2O     atmosphere/photosynthesis
          Potential Products
• Fuels
  • H2 hydrogen
  • CH4 methane
  • CH3CH2OH ethanol
            Potential Products
• Natural products (complex synthesis)
  •   Vitamins
  •   Therapeutic agents
  •   Pigments
  •   Amino acids
  •   Viscosifiers
  •   Industrial enzymes
  •   PHAs (biodegradable plastics)
          Potential Products
• Engineered products
  • Starting materials for polymers (such as
    rubber, plastics, fabrics)
  • Specialty chemicals (chiral)
  • Bulk chemicals (C4 acids)
           Problem to Solve
• 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 niches
• Optimized parameters
  • Low nutrients
  • Defense systems
                 Opportunity
Redesign bacteria with industrially-valuable
 parameters optimized
  • Redirect metabolism to
     specific products
  • Increase metabolic efficiency
  • Increase 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 cell

• Must redesign the
  network
  • Understand the
    connections to achieve
    end result
               What’s New?
• Genomics
  • Bacterial genomes small (1000 = human)
  • Hundreds of bacterial genome sequences
    available
  • Provides the blueprint for the organism (the parts
    list)




             New platform for redesign
            What’s New?
• Increased 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
  advantage
• Cannot 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 pieces




 Strategy:
    Understand the modules and their connections
    Redesign in blocks
      Methanol as an Alternative
           Biofeedstock
•   Soluble in water
•   Inexpensive            CH3OH
•   Pure substrate
•   Bacteria that use it   chemicals
      well-studied
Methylotrophic Bacteria
     CH3OH (methanol)
              O2




       CO2, H2O, cells
     Specified product
                 Approach

• Define functional                                   methanol
  modules by                                      MEDH
                                              CH OH
                                              3   cyt
                                                    c
                                                    L
                                                      HCHO
                                                           MADH
                                                               3 2 NH
                                                               CH
                                                           amicyanin

                                                    H4F


  experimental and
                                                                H4MPT
                                        Assimilation      HCHO               Dissimilation
                                                Methylene H4F      Methylene H4MPT
                                                                         NADH
                                CO2             NADPH Methenyl NADPH
                                                                 H4F
                                      Serine                           Methenyl H4MPT




  evolutionary analysis
                                       cycle


                                  C3 Compounds
                                                   x
                                                   BIOMASS
                                             Purines
                                                        N10-Formyl H4F
                                                             ATP
                                                        Formate
                                                                             N5-Formyl H4MPT

                                                                                     Formyl MFR
                                                             NADH
                                             fMet-tRNA
                                                                                    2H
                                                       CO2                   CO2         CO2




•Manipulate modules
to optimize product                                    product

 •Optimize process parameters
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
                         CH3OH



 Biosynthesis                          Energy metabolism
 (assimilation)     C3                 (dissimilation)
                                 CO2

                  Biomass
                                 PHA (biodegradable
                                 plastic)
Methylotrophic Metabolic Modules
                                                     Methanol

                                       Methanol
                                       Oxidation
        PHA
          PHA
          cycle                                      Formaldehyde
Glyoxylate
                       Serine         Methylene H4F
Regeneration
                       cycle
cycle


               TCA                                           H4MPT-linked
                                H4F-linked
               cycle                                         C1 transfer
                                C1 transfer


                                                   Formate
    CELLS
                                              FDH1           FDH2           FDH3



                                                      CO2
             Constraints
• Understanding how the system is
  integrated in time and space
• Changing how it works
           Work in Progress
• Use genome-wide
  techniques to assess
  expression of genes
  within each module
• Use metabolic modeling
  to make predictions about
  flow through each module
                                        BIOMASS

• Use labeling techniques                         CO2

  to measure flow through
  each module

  Results: redesign the metabolic network to overproduce a
  biodegradable plastic
                                                                                         Multi-tiered datasets
 Metabolite pools
 Xiaofeng Guo
                            Average spectra for serine peak
          Abundance                             156
              400

              360
                                                                                 228
              320




                                                                                                                                                                                                                                                        microarrays: mRNA
              280

              240

              200

              160

              120

               80

               40

                0
                    10 1




                    100
                           11 4




                              120
                                  12 8
                                         13 8




                                          140   160
                                                      17 4




                                                         180
                                                             18 4




                                                                    200
                                                                          22 0



                                                                          220
                                                                                  24 2



                                                                                  240
                                                                                         256
                                                                                         260
                                                                                                                                                                                                                                                        Yoko Okubo, Betsy Skovran
                                                  m/z




Enzyme activities
Xiaofeng Guo
                              CH3OH                                                              Fluxes
                  H4 F
                            MDH

                             HCHO
                                   H2O, 2e-
                                          H4MPT
                                            Fae
                                                                                                 Chris Marx                                                                                                                                              proteomics: proteins
       Methylene
                          spont.
                       H2 O
                                     H2 O
                                                Methylene                                        Steve Van Dien                                                                                                                                          Julia Vorholt group
          H4F CH2=H4F                   CH2=H4MPT MPT
                                                 H4
Serine
            NADPH
                       MtdA          NAD(P)H MtdA, MtdB                                          Greg Crowther                                                                                                                                           Murray Hackett group
                CH=H4F                      CH=H4MPT                                                                  2 NADPH




           H2 O                       H2 O      Mch
                                                                                                     G6P
                                                                                                                      0
                                                                                                                                           R5P
                                                                                                                                              PP Pathway                                           Biomass yield: 4.98
                       Fch                                                                         0.29

                                                                                                      F6P
                                                                                                                  0.01

                                                                                                                             E4P
                                                                                                                                                                       NADH



                                         CHO-H4MPT                                                 0.30
                                                                                                                                                    -KG
                                                                                                                                                                   0
                                                                                                                                                                                Succ-CoA



                 CHO-H4F
                 H2 O               H2 O                                                            Triose-P               0.03
                                                                                                                                             0.09

                                                                                                                                              Citrate      TCA
                                                                                                                                                                                          0.04
                                                                                                                                                                                                         1.00
                                                                                                                                                                                                                    Propionyl-CoA

                           FtfL H4MPT
                                                                                                                                                                                   Succinate
                                                                                                   0.35                                                    Cycle
                                                                                                                       0          Ac-CoA            0.09                                                                                   Acetyl-CoA
                                                                                                     3-PG                                                                          1.00



           H4F, ATP                         Fhc                                                                Pyruvate
                                                                                                                                     0                             Malate
                                                                                                                                                                            3.27
                                                                                                                                                                                     Malyl-CoA
                                                                                                                                                                                                                2 NADH
                                                                                                                                                                                                                FADH2
                                                                                                                                                                                                                               1.00
                                                                                                                                                                                                                                           Conversion
                                                                                                                                                                                                                                           Pathway

                             HCOOH                                                                 0.35         0.21                              OAA      2.27
                                                                                                                                    2.56                                                         3.27
                                                                                                               2.83
                                                                                                                            PEP                                                                                         Butyryl-CoA
                                                                                                     2-PG
                                                                                                                                              Serine Cycle                   Glyoxylate Ac-CoA

                                 FDHs                                                             CO2
                                                                                                               3.17
                                                                                                                           Serine
                                                                                                                                    CO2

                                                                                                                                                                  Glycine
                                                                                                                                                                                     3.27

                                                                                                                                                                                          NADPH
                                                                                                                                                                                                                 NADPH
                                                                                                                                                                                                                               1.00

                                                                                                 NADPH         0.62                                                                                     2.92       Hydroxybutyryl-CoA
                                                                                                  2 e-

                        NADH CO2                                                                  CO2
                                                                                                 NADH
                                                                                                               5.55
                                                                                                                      Methylene-H4MPT
                                                                                                                                  6.17
                                                                                                                                                            3.21


                                                                                                                                                                                      NADPH
                                                                                                                                                                                                                               0.46


                                                                                                                                           3.84                          0.62                                              PHB
                                                                                                  2 e-                      HCHO                    Methylene-H4F                     ATP
                                                                                                                                                                                      Formate                                          0
                                                                                                                                                     Cell membrane                                                       NADH                 4 H+ext
                                                                                                               10.00                                                                             0.56
                                                                                                  CH3OH                     HCHO                                                                                                      19.3
                                                                                                                                                                                                                         2 H+ext              ATP
                                                                                                                                                                                   CO2       NADH
                                                                                                                      2e-
               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 subpopulations
• Difficult 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 (10-15)
• Number of DNA molecules ~2-3
• Number of mRNA molecules for a specific
  gene ~10-10,000
• Total protein amount ~amoles (10-18)
• Total moles of specific metabolites ~ amoles
  (10-18)
• Respiration rates ~fmol/min/cell (10-15 )
         New Interdisciplinary
            Approaches
• Combine
  •   Genomics
  •   Computational biology
  •   MEMS (microelectromechanical systems)
  •   Systems integration
  •   Nanotechnology
     Microscale Life Sciences Center
        University of Washington
• Center of Excellence of Genomic Sciences funded by NIH
  NHGRI
• 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
           Microsystem-Based Devices for
                Studying Single Cells
            •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)


 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)
               Evidence for Heterogeneity
       • Single-cell cell cycle analysis: growth
                                                        Single Cell Division Times During MeOH Growth

                                  5                                                                                                 Range:
                                 4.5

                                  4                                                                                                 2.5-4.3 hr
                                 3.5
                    Time (hrs)

                                  3

                                 2.5

                                  2

                                 1.5

                                  1

                                 0.5

                                  0
                                       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




                                                                           Single Cell Division Times
                                            12

                                            10

                                            8
                                  # cells
                                            6

                                            4
Tim Strovas,                                2

Linda Sauter                                0
                                                 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
                                                                              Time, Hr
               Summary

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

								
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