Bacteria

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Bacteria

• Single cells
• Small size (1-5 mm)
• Rapid reproduction
• Genomic and genetic capabilities

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Bacterial Diversity

• 4 billion years of evolution
• Ability to thrive in extreme environments
• Use nutrients unavailable to other organisms
• Tremendous catalytic potential

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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
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Bacteria as Factories
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Harnessing Catalytic Potential of Bacteria

• Use bacteria as self-replicating multistage
  catalysts for chemical production
• Environmentally benign
• Renewable starting materials (feedstocks)

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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/photosy
  nthesis

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Potential Products

• Fuels
• H2 hydrogen
• CH4 methane
• CH3OH methanol
• CH3CH2OH ethanol

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Potential Products

• Natural products (complex synthesis)
• Vitamins
• Therapeutic agents
• Pigments
• Amino acids
• Viscosifiers
• Industrial enzymes
• PHAs (biodegradable plastics)

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Potential Products

• Engineered products
• Starting materials for polymers (such as rubber,
  plastics, fabrics)
• Specialty chemicals (chiral)
• Bulk chemicals (C4 acids)

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Problem to Solve

• If bacteria are such wonderful alternatives, why
  are our chemicals still made from environmentally
  hazardous feedstocks?

Bacterial processes are too expensive
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Natures Design Solutions

• Competitive advantage in natural niches
• Optimized parameters
• Low nutrients
• Defense systems

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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?
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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

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Whats 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
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Whats New?

• Increased understanding of how new kinds of
  metabolism arose

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Changing Environmental Niches
Selection for novel metabolic capabilities
Time before present
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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?

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Modular Aspect of Metabolism

• Metabolic capabilities were built in blocks, like
  puzzle pieces

Strategy Understand the modules and their
connections Redesign in blocks
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Methanol as an Alternative Biofeedstock

• Soluble in water
• Inexpensive CH3OH
• Pure substrate
• Bacteria that use it chemicals
• well-studied

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Methylotrophic Bacteria
CH3OH (methanol)
O2
CO2, H2O, cells
Specified product
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Methylobacterium extorquens AM1

• Grows on one-carbon compounds (methanol,
  methylamine)
• Also grows on multi-carbon compounds (succinate,
  pyruvate)
• Substantial toolkit for genetic analyses
• Genome sequence (with UW HGC)
• Plant symbiont

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Approach
methanol

• Define functional modules by experimental and
  evolutionary analysis

• Optimize process parameters

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Methylotrophy
CH3-X
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Approach and Results

• Identify the components
• Identify the genes and enzymes
• Determine their function
• Results
• Identified over 100 genes
• Generated mutants in each
• Analyzed which functions are missing
• Growth
• Enzyme activities
• Measure cofactors
• Study expression of genes

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Methylotrophic Metabolic Modules
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Methylotrophic Metabolic Modules
Methanol
PHA
Formaldehyde
Methylene H4F
Formate
CO2
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Constraints

• Understanding how the system is integrated in
  time and space
• Changing how it works

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Work in Progress gene expression

• Use genome-wide techniques to assess expression
  of genes within each module

DNA expression microarrays
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Expression Microarrays (DNA Chips)

• Design a segment of DNA complementary to a small
  stretch of every gene in the genome
• Specific to that gene
• Can be used to detect that gene
• Spot a sample of these DNA molecules in a very
  small spot (usually on a microscope
  slide)--common to have 10,000 spots/slide

ATGGCTTAAAGATCCCATGGCTA
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Expression Microarrays (DNA Chips)

• Extract RNA from cells, make a DNA copy (cDNA),
  label with a fluorescent dye
• Condition 1 label green
• Condition 2 label red
• Mix, hybridize to the slide
• Each mRNA fragment only binds to the spot
  containing the gene
• If no change in expression yellow
• If expression went up in Condition 1 green
• If expression went up in Condition 2 red

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Expression Microarrays (DNA Chips)

• If no change in expression yellow
• If expression went up in Condition 1 green
• If expression went up in Condition 2 red
• If expression is below the detection limit, no
  color
• Results reported as fold change (difference)

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Work in Progress proteomics

• Separate all proteins in cell by size and then by
  charge
• Cut out samples (spots), generate a mass pattern
  (mass spectrometry)
• Use mass to predict peptide
• Search genome to identify
• Can compare with the same conditions as the
  microarray

• Use genome-wide techniques to assess expression
  of proteins within each module

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Work in Progress Flux Analysis

• Use flux-balance model (Palsson)
• Mass balance equation for each reaction
• Use genome sequence to deduce metabolic pathways
• Use optimization techniques to solve for biomass
  production
• Problem underdetermined

• Confirm model with 13C-labeling
• Steady-state labeling with 13C-substrate
  (chemostat)
• Measure isotoper distribution for amino acids
• Deduce fluxes

S. Van Dien
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Work in Progress overview

• Use genome-wide techniques to assess expression
  of genes within each module
• Microarrays mRNA
• Proteomics proteins
• Use flux-based techniques to understand how the
  pathways work
• Metabolic modeling predictions about flow
  through each module
• Labeling techniques measure flow through each
  module

Results redesign the metabolic network to
overproduce a biodegradable plastic
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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