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Sunday, 09 September 2018 21:57

Photon Factors Favor Fancy Fuels - BacterioFiles 355

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Published in Bacteriofiles

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: 

bf355yeastMicrobe 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

Last modified on Sunday, 09 September 2018 22:03
Jesse Noar

Jesse Noar is microbiologist with a PhD from North Carolina State University and Bachelor's from Cornell. Most of his research has focused on the amazing abilities and potential uses of bacteria, especially those found in soil. Jesse hosts the BacterioFiles podcast highlighting the most interesting recent microbiology research on all kinds of different microbes, part of the ASM family of podcasts. Learn more at asm.org/bacteriofiles or at www.bacteriofiles.com.

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