18. Biofuels II -> biofuels and biochemicals

Production of acetone using Clostridium

  • Produced in Clostridium acetobutylicum

  • Ferments sugars to acetone, butanol and ethanol (ABE process)

  • Natural process under anaerobic conditions

  • Ethanol, acetone and butanol secreted from the cell (reduces costs)

This process could not compete with production from petroleum after WW2

Over 6,000 items are currently produced from oil

We are going to have to find renewable, low cost, alternatives within the next hundred years (preferably sooner)

What are the major issues in using microbes to produce chemicals

A. Cost

B. Genetically engineering a strain that stably produces high amounts of the chemical

C. Mutation of genetically engineering strains

D. Scaling up processes to industrial scale

E. All of the above

Microbial production of organic and amino acids

  • For some compounds, microbial production has always been the optimal route (organic acids, amino acids, vitamins)

Production occurs at huge scale

  • Well established processes

  • MSG sells for about £5-10 a kg on amazon (low to medium value product)

  • All amino acids are industrially produced via natural cellular processes

Genetically engineering organisms for production of novel compounds

  • Only a few products are naturally produced by microbes in quantities sufficient for commercialisation

  • For synthesis of novel compounds or to increase production (and secretion) of novel compounds organisms have to be genetically engineered

  • Characterisation of the appropriate biosynthetic pathway is required for production of novel compounds

What properties are important when choosing a species for production of chemicals

A. Fast growth

B. Amenability to genetic manipulation

C. Production of precursors for the desired chemical

D. Ability to export chemicals from the cell

E. All of the above

Production of diesel in Escherichia coli

  • Expressed the pathway in E. coli

  • Claimed that hydrocarbons were secreted into the media

  • Were adding a small amount of detergent that was lysing cells

  • Built a pilot plant

  • Process wasn’t commercial

  • Company sold in 2013

Introduced a pathway for production of branched fatty acyl-ACPs

Introduced the cyanobacterial hydrocarbon biosynthesis pathway Hydrocarbons were not secreted into the media

Producing hydrocarbons similar to petrol in Escherichia coli

  • Modified an enzyme that produces smaller fatty acids

  • Alkanes ranged from C8 to C16

  • Smaller alkanes were secreted from the cell

  • The final engineered strain produced up to 580.8 mg l(-1) of SCAs consisting of nonane (327.8 mg l(-1)), dodecane (136.5 mg l(-1)), tridecane (64.8 mg l(-1)), 2-methyl-dodecane (42.8 mg l(-1)) and tetradecane (8.9 mg l(-1))

Producing artemisinin in yeast - the great synthetic biology success story

  • Artemisinin is an anti-malarial drug derived from the plant Artemisia annua

  • Plant derived artemisinin fluctuated in price and production varied hugely from year to year

  • Used synthetic biology to express the artemisinic acid pathway in Saccharomyces cerevisiae

    • Up-regulated production of the precursor-farnesyl pyrophosphate (FPP)

    • Introduced Four novel genes for production of artemisinic acid

    • Artemisinic acid secreted from the cell

  • Yields are now at 25 g per litre (small-scale batch process)

  • Production costs are between $US350-400 per kg

  • Can’t compete with plant derived artemisinin (the price stabilised after this process was developed)

  • Production plant was sold by Sanofi

Adapting this process for production of farnesene

  • Farnesene is a low value hydrocarbon which can be used as a biofuel or chemical feedstock

  • Single enzyme required for production of farnesene from farnesyl pyrophosphate

    • Enzymes introduced into yeast to increase production of farnesyl pyrophosphate (FPP)

    • Farnesene synthase introduced into yeast for production of farnesene

Farnesene is a low value chemical

  • Farnesene is a low value (~US$3 a kg) hydrocarbon which can be used as a biofuel or chemical feedstock

  • To be commercial, production has to occur at large scale and be robust

    • Built an expensive plant (40 million liter capacity)

    • Brazilian sugar cane was used as the feedstock

    • Similar process as used for producing ethanol in yeast except the process does not rely on fermentation

One billion dollars later

  • Predicted production of 9 million litres in 2011, 44-50 million litres in 2012

  • This was reduced to 1-2 million litres in 2011. Even this target wasn’t met

  • The plant was sold for US$96 million in 2017 (with IP)

  • Now focusing on ‘high value’ chemicals

Investors are still not impressed → Investors have lost over 1 US billion dollars

What are the major issues with genetically engineering an organism to produce large amounts of chemicals

A. Time taken to engineer the organism

B. Diverts energy away from growth

C. May toxify the media

D. May not be scalable

What went wrong?

  • The company has not divulged what went wrong (despite receiving public money)

  • Two likely possibilities

  • Upscaling

  • Mutation of strains

  • Upscaling problems include mixing, aeration, cleaning, contamination, reusing yeast

Evolution will always win

A scientific man ought to have no wishes, no affections, - a mere heart of stone Charles Darwin

  • Diverting energy and resources from growth towards production of a specific compound is not to the organism’s advantage

  • Therefore your engineered strain is less fit

  • Any mutant that rediverts energy and resources from production of a specific compound back towards growth will have a selective advantage

Which genetic mutation is likely to be more stable?

A. Deletion of a native gene

B. Over-expression of a foreign or native gene

Inactivating genes is relatively easy for microbes

  • Deletion of genes is a relatively stable mutation (Harder to find a substitute gene to compensate for its loss)

  • Relatively easy to inactivate a gene (mutation in an essential amino acid)

A similar situation happened with cyanobacterial biotechnology

  • Company went bankrupt in 2017. Investors lost over 200 US million dollars

Re-calibrating the field

  • Failures re-focused the field

  • Focus on higher value products

  • Improving production methods

  • Better strains

  • Unique products

High vs low value compounds

  • All ‘viable’ companies are focusing on ‘high value’ compounds

    • Nutraceuticals

    • Pharmaceuticals

    • Specialty chemicals

Higher value compounds can be produced using lower volume batch cultures

  • Solves two issues

    • Upscaling → from lab scale to bioreactors with 10s to 100s of litres is not as challenging

    • Genetic mutation is not as great an issue

      • Can start with fresh starter cultures

      • Cheaper to clean out bioreactors

      • Shorter culture periods

      • Less cells

  • Success is dependent on value of products

    • Artemisinin was ~US$350-400 a kg

Making unique specialist products is the best option

  • Spider silk is tougher than steel or Kevlar BUT you can’t farm spiders

    • Using E. coli to make spider silk

    • Spinning artificial spider silk

Maybe focus on really, really high value products

  • Sea silk (byssus)- the most valuable fabric in human history

  • Genesis 41:42

    And Pharaoh took off his ring from his hand, and put it on Joseph's hand, and arrayed him in clothes of byssus, and put a gold chain on his neck.

  • Reportedly, a Japanese businessman offered US$3 million for a square of fibre of 18 inches per side