Monday, September 10, 2018

BacterioFiles 355 - Photon Factors Favor Fancy Fuels

Saccharomyces cerevisiae yeast
This episode: Engineering yeast to control their metabolism using light and dark for the production of advanced biofuels and chemicals!

Download Episode (16.1 MB, 17.7 minutes)

Show notes:
Microbe of the episode: Equine arteritis virus

News item

Journal Paper:
Zhao EM, Zhang Y, Mehl J, Park H, Lalwani MA, Toettcher JE, Avalos JL. 2018. Optogenetic regulation of engineered cellular metabolism for microbial chemical production. Nature 555:683–687.

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Episode outline:
  • Background: Lots of promise in using microbes as tiny factories
    • Can transform lots of things into lots of other things
    • Medicine, fuels, other useful chemicals
  • With high value products like drugs, don't need too much efficiency
    • But fuels and such, need to optimize
    • Reduce cost of inputs, maximize outputs
  • Essential to balance metabolism
    • So intermediates don't build up and cause bottleneck or side paths or toxicity
    • Tight control required, but tricky to achieve
  • Can set up so cells regulate engineered metabolism automatically
    • Not always ideal; requires different steps simultaneously
  • Alternative is having multiple steps
    • Build up precursors in one step, then change to next to convert
    • Helpful to make inducible, eg by chemical addition or temperature or something
  • What’s new: Now, scientists publishing in Nature have developed a system in yeast for producing valuable chemicals by controlling the cells with light!
  • Light is cheap and compatible with most processes
    • Easy to add or remove
  • Methods: System called optogenetics
    • Uses blue light-sensitive transcription factor from microbe Erythrobacter litoralis
      • Aerobic, marine phototroph
    • And promoter that protein activates expression from, then put whatever gene
  • Tried GFP first to test
    • Could get 43x more fluorescence with constant light than dark
    • Or less, if desired, with pulses
    • Similar expression to common constitutive promoter ADH1 - maximized
  • Then on to application
    • Yeast growing on sugar – two pathways after glycolysis
    • Convert to ethanol if too much – fermentation
    • Otherwise respiration – break all the way down to CO2
  • Here wanted other products, so needed to inhibit ethanol production
    • Used optogenetic process to control
    • Set up pyruvate decarboxylation control with light
      • Takes glycolysis product and directs to growth/ethanol
    • Here, can turn on with light, turn off without
    • So when growing yeast, turn on so they can grow well
      • Then move to dark vessel to make product
  • Worked out well – strain could grow ~90% of normal rate in light
    • If grown with ethanol/glycerol, didn't need light – independent of this enzyme
  • Then optimize for desired product
    • Here, lactate or isobutanol
      • Former: precursor chemical and such, easier to test, low toxicity. 
      • Latter also good chemical, and drop-in biofuel
    • Combine two different light mechanisms – one to shut off and one to turn on
      • For shutting off, have light activate protein that shuts off another protein
  • Lactate worked well, production increased
  • Isobutanol – multiple enzymes in pathway but only regulated first
    • Others produced constitutively, but no precursors to act one
    • Tried varying amounts of light and dark culturing and cell density when switching to max
    • More cells seems better, but only to a point; too many and they get unhealthy, can't produce
    • Gave 2% glucose – 20 grams in a liter of medium
      • Got up to 34 milligrams isobutanol per gram glucose
        • Theoretical max of ethanol is 511 mg, usually more like half that
        • But butanol has 4 carbons instead of 2, so expect more like 100mg/g
    • Then tried 15% glucose, got 8 mg/g; less glucose consumed, metabolism stalled
  • Thought maybe cell energy (electron carriers) ran out in dark; so pulsed light to recharge a bit
    • Keep metabolism going
    • Got production up to 22 mg/g, along with other good biofuel byproduct
    • Still less yield than with 2% glucose, but much higher final concentration
  • Then tried in fermenters, with dark phase a fed-batch
    • instead of all glucose at once, pump in gradually over time as needed
    • Got up to 53mg/g isobutanol and 14 mg/g other byproduct
  • Summary: Engineering yeast to control their metabolism with light exposure, can greatly increase their ability to produce isobutanol biofuel instead of ethanol, more cells, or other products
  • Applications and implications: Butanol and other advanced biofuels important to develop
    • Ethanol is valuable as gas additive; cleaner, cheaper, higher octane
      • But only works up to certain concentration, only in certain engines (and only gas)
      • Also lower energy density
    • Butanol can replaced gas directly, works in all gas engines
      • And other compounds also good
      • And other engines need other fuels, like jets/diesel/ships
    • So developing tech to produce them commercially is important
  • What do I think: Cells in nature optimize their own metabolic regulation
    • In nature, turn on best enzymes at right times/levels to maximize survival in environment
  • Not always best for what we want them to do
    • Can modify environment to encourage other production
    • But for optimum production, add/modify enzymes
    • And even better, add ability to regulate easily, like here
  • Microbes' cellular chemistry will be more and more important for sustainable production

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