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