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JBEI Researchers Engineer Yeast to Produce n-Butanol

6 December 2008

Jbeiyeast
n-butanol production by the different strains. Click to enlarge.

Researchers at the Joint BioEnergy Institute (JBEI), led by Dr. Jay Keasling at UC Berkeley, have engineered the common industrial yeast Saccharomyces cerevisiae with an n-butanol biosynthetic pathway, resulting in a ten-fold improvement in n-butanol production from one of the strains to 2.5 mg/L. An open access paper on their work was published online 3 December in the journal Microbial Cell Factories.

Butanol has a number of advantages over ethanol for use as a biofuel—it is more hydrophobic; has a higher energy density; can be transported through existing pipeline infrastructure; and can be mixed with gasoline at any ratio.

A variety of Clostridial species can produce n-butanol fermentatively. However, the team notes in their paper, Clostridia are not ideal because of the relative lack of genetic tools to manipulate their metabolism, their slow growth, their intolerance to n-butanol above 1-2% and oxygen, and their production of butyrate, acetone, and ethanol as byproducts.

Recently, two groups have re-constructed the n-butanol pathway from Clostridia and measured n-butanol production in Escherichia coli (~1 mM). We chose Saccharomyces cerevisiae as a host for n-butanol production because it is a genetically tractable, well-characterized organism, the current industrial strain alcohol (ethanol) producer, and it has been previously manipulated to produce other heterologous metabolites. Since n-butanol and ethanol only differ by two carbons, S. cerevisiae may be able to tolerate high concentrations of n-butanol by the same mechanisms it tolerates ethanol.

—Steen et al. 2008

The researchers used n-butanol pathway isozymes from a range of organisms, and engineered a number of different strains of S. cerevisiae. Results varied, but the best strain, ESY7, produced 2.5 mg/L n-butanol from 2% galactose as a carbon source.

Comparison of our strain to the native n-butanol producers, Clostridia (~10 g/L), or the recently engineered E. coli strains (~0.5 g/L) provides a goal for future n-butanol titer. Given the results presented above and S. cerevisiae’s other attributes—inherent tolerance to solvents, widespread use for industrial production of ethanol, and ability to withstand oxygen (as opposed to Clostridia)—S. cerevisiae may be an ideal host for industrial n-butanol production. While increases in product titer will certainly be necessary, increases of this magnitude and greater have precedence.

—Steen et al. 2008

Keasling has financial interests in Amyris and LS9, both of which are involved in developing advanced renewable hydrocarbon fuels.

The Joint BioEnergy Institute (JBEI), one of three new US Department of Energy Bioenergy Research Centers, is a San Francisco Bay Area scientific partnership led by Lawrence Berkeley National Laboratory (Berkeley Lab) and including the Sandia National Laboratories (Sandia), the University of California (UC) campuses of Berkeley and Davis, the Carnegie Institution for Science and the Lawrence Livermore National Laboratory (LLNL). JBEI’s primary scientific mission is to advance the development of the next generation of biofuels.

Resources

  • Eric J. Steen, Rossana Chan, Nilu Prasad, Samuel Myers, Christopher J. Petzold, Alyssa Redding, Mario Ouellet, Jay D. Keasling (2008) Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol. Microbial Cell Factories 7:36 doi: doi:10.1186/1475-2859-7-36

December 6, 2008 in Biobutanol, Biotech | Permalink | Comments (18) | TrackBack (0)

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Comments

Butanol may be the liquid fuel of the future. It is a liquid at ordinary temperatures and pressures. It has almost the energy density of gasoline per unit weight and volume.

It is a single well known chemical and engines can be optimised to burn it. It does not evaporate quickly, so it is less of a fire hazzard, and it does not degrade as rapidly with time as gasoline and could last for a hundred years.

Vapore-jet and MSR should modify their vaporizer stove to burn it as it is much safer than white gasoline. A mantel lantern that burns it would be nice as well.

It may be able to be used also in diesel engines and certainly as jet fuel with few modifications, and its long life allows a large ready reserve to be established. It may be considered a permanent fuel and bottled for use in small engines.

There is no reason that it cannot be made from Natural gas and perhaps natural gas could be part of the nutrition for the yeasts. Its chemical production will irritate many people but it is still the same chemical.

It could be considered as the best fuel to be be left in the tank of Plug-In-Hybrid vehicles that run mostly on batteries most of the time. With the universal availability of small and very small engines, and the universal high price of large Lithium batteries, every car with electric drive should have at least a very small fuel powered charger. ..HG..

How are they going to keep the n-butanol producing yeast from mixing with standard yeast cultures? Do you want your beer or bread yeast producing a chemical that "will irritate many people"? We are children playing with matches when it comes to changing food crop genomes to make toxic fuels. Will it take a major event to get us to reconsider our careless approach to the types of organisms that we modify. Let's hope that the first genetically modified incident to teach us that we should have been more careful is not an unimaginable catastrophy.

They can do all because they think they can.

Virgil

Roman epic poet (70 BC - 19 BC)

For all the green naysayers out there, if this new organism contaminates beer yeast it will cure alcoholism.

Think positive.

Keeping genetically modified corn seed out of a corn field is rather difficult, but keeping any 'foreign' yeast organism out of a brewery is not difficult.
Testing for the presence of butanol in beer or other food is very easy. The toxicity of butanol is very low compared to many other biological toxins that may be present in 'contaminated' products.

James

I understand your concerns but they are unfounded in that particular case: these finely tuned yeast are so specialized that they are not very robust and can't survive out of the environment they have been designed for so don't worry about your beer.

My personal concern is that I still need to be convinced that these finally tuned yeast or bacteria can be used in an industrial environment aiming at huge volume of production. I haven't seen any evidence of this here or there, their use seems extremely technical and tricky,

Good News at last!!!
My brother wants to brew some fuel right now..
We won't need the gas station anymore.
Thank the Lord for technology.

@ James White: please refrain from commenting anymore- critical thought is just not your thing, ok?

2.5 mg/L? That's a hefty 0.00025%! And from only 2% sugar.

We note that the specific form of butanol is n-, i.e. with the four carbon atoms lined up in a straight chain.

Perhaps it would be better if one could produce isobutanol, or as a one versed in chemistry would put it, methylpropanol. My reason: methyl-isopropanol might have a higher octane rating.

Somebody mentioned possible toxicity of butanol. I hope it isn't nearly as bad as methanol. During the prohibition era in the US (1919 to 1933), many people went blind because poorly processed booze was contaminated with "wood" (i.e, methyl) alcohol.

As described above the concentrations of butanol achieved are very low. Yeast have been selected over hundreds if not thousands of years to produce ethanol... and today can tolerate 10-15% ethanol titers. The question is: can we genetically modify yeast to produce butanol in similar concentrations (which we will need to do if we ever plan to retrofit existing ethanol facilities and use the infrastructure that's already been built). Some butanol startups (Gevo for example) already claim to be able to produce butanol in the 10% range.

I've been hearing people talk about butanol's toxicity for some time and this thread inspired me to finally compare them. It took a little digging, but looking at the MSDS data available through Oxford University (http://msds.chem.ox.ac.uk/#MSDS) and comparing n-butanol with n-heptane (the primary component of gasoline) I did find a similar base for comparison.

n-butanol IVN-MUS LD50 = 377 mg/kg
n-heptane IVN-MUS LD50 = 222 mg/kg

Meaning that butanol, at least by this crude comparison is about 1.7 times as toxic as gasoline.

Looking at it in more concrete terms, applying additional butanol toxicity data (ORL-RAT LD50 = 790 mg/kg) and making the jump that a rat model is reprentative of human mortality... a little more than two shot glasses (100 ml total) of butanol would kill 50% of people weighing 100 kgs (220 lbs). If you want to check my math you'll want to know that the specific gravity of butanol is ~ 0.8.

Sam Nejame
Promotum

Actually the butanol is less toxic than heptane.

The LD50 is the amount of a substance in grams that results in 50% mortality. Since the LD50 of butanol is larger than heptane, it is actually less toxic. Another way to look at it is that more butanol is required to achieve the same number of dead rats.

Actually even if butanol were an outright poison, I would regard it as an intellectual frustration for us not to utilize butanol only for that reason. The solution to the problem of toxicity is simple: Keep it out of reach of children, and take precautions to keep both liquid and vapor out of your system.

This is true not only of butanol but also, ethanol and methanol as well. One is an addictive drug, the other is just plain poisonous. But again, I would consider it a frustration to give up either, or both, simply because they are not harmless to have in one's body.

hah awesome thanks
http://www.carshowsdate.com/

Thanks Jim, I could't understand that either.
Poisons are a problem for techs, supply chain etc, better handled these days but as bute is better according to Sams figures , that wouldnt be a bigger issue.

Conclusion:

100 ml of butanol or 150 ml of gasoline can kill about 50%.

How many died from drinking gasoline or butanol lately?

There are 100+ more toxic liquids around.

Since butanol is a lot less evaporative than ethnaol and gasoline, kids would not sniff it as much. As far as drinking it, a bad taste could be added.

We actually use butanol in our biotechnology lab from time to time to extract certain kinds compounds from water-based solutions. It doesn't smell much, and we don't use any special precautions except the standard precautions of labcoat, gloves and safety glasses.

This is some interesting information from the Dow Chemical website. Surprisingly, it may be much better for the environment than gasoline as it is acutely non-toxic to the marine environment and biodegradable.


Product Overview

* n-Butanol (normal butanol) is a clear, colorless liquid that is flammable. It has a characteristic banana-like odor1and is used to produce other chemicals, as an ingredient in formulated products such as cosmetics, and as a solvent.

* n-Butanol demonstrates an overall low order of toxicity.3 Acute (24 hours or less in duration) overexposures may cause irritation to the eyes and skin or can be harmful if inhaled. Prolonged (greater than 24 hours), excessive exposure to vapors may cause serious adverse effects, and even death. Birth defects have been observed in animals exposed to high concentrations of n-butanol which also caused serious adverse effects to the exposed mothers.

* Occupational and consumer exposure is dependent upon the conditions under which n-butanol or the end-product is used.

* n-Butanol is practically non-toxic to aquatic organisms and birds on an acute basis. The material is readily biodegradable.


Jim you're right... Doh! It takes more butanol than heptane to achieve 50% mortality... Good thing I'm not a health and safety officer...

Along that line, it's interesting to note that heptane is only the major component of gasoline... there are ~500 different species... and many of them like benzene are bad actors and carcinogens... So, if this LD50 data is truly representative, than butanol is definitely less toxic.

Sam Nejame
Promotum

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