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Researchers Engineer Bacteria to Produce Nonnatural Alcohols with Higher Energy Density

Liaoc6
Schematic representation of the biosynthetic pathway of the 6-carbon alcohol 3-methyl-1-pentanol. The engineered nonnatural metabolic pathway is shaded in lavender. Click to enlarge. Credit: PNAS

Researchers at UCLA have developed a nonnatural biosynthetic pathway enabling the bacteria Escherichia coli to produce various long-chain alcohols with carbon numbers ranging from 5 to 8. Higher carbon alcohols are attractive biofuel targets because they have higher energy density and lower water solubility. By way of comparison, ethanol has two carbons; butanol has four.

To demonstrate the feasibility of their approach, they optimized the biosynthesis of a 6-carbon alcohol: 3-methyl-1-pentanol. A paper on the work by Dr. James Liao and colleagues was published online 8 December in the Proceedings of the National Academy of Sciences.

In January 2008, Liao reported the genetic modification of E. coli for the efficient production of several higher-chain alcohols including isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol. (Earlier post.) Instead of relying on fermentation for the production of the alcohols, Liao leveraged E. coli’s highly active amino acid biosynthetic pathway by shifting part of it (its 2-keto acid intermediates) to alcohol synthesis. Gevo, a biofuels startup, licensed that technology, and Liao joined Gevo’s scientific advisory board.

Nature uses a limited set of metabolites such as organic acids, amino acids, nucleotides, lipids and sugars as building blocks for biosynthesis. These chemicals support the biological functions of all organisms. So far, construction of artificial biological systems is limited by the existing metabolic capabilities. By supplying living cells with chemically synthesized nonnatural amino acids and sugars as new building blocks, it is possible to introduce novel physical and chemical properties into biological entities.

These efforts raise an interesting question: Can we rewire metabolism in a bottom-up fashion to produce nonnatural metabolites from simple carbon source? If so, such engineered artificial metabolism should be able to expand the chemical repertoire that living systems can use and produce. To begin to address this question, we developed a strategy to produce 7-(C7) to 9-carbon (C9) 2-keto acids, which can lead to useful nonnatural alcohols (C6–C8).

—Zhang et al. 2008

In this work, Liao’ team first used the existing metabolic capability of E. coli to synthesize 2-keto-3-methylvalerate, the 2-keto acid precursor of amino acid Lisoleucine. The chemical structure of 2-keto-3-methylvalerate is similar to that of 2-ketoisovalerate, which is converted to a butanol precursor via chain elongation (increasing its carbon content) by a set of enzymes. The researchers reasoned the enzymes (LeuA, LeuB, LeuC and LeuD) might be promiscuous enough to take 2-keto-3-methylvalerate through the same elongation cycle and produce a novel compound: 2-keto-4-methylhexanoate.

They then speculated that the 2-keto-4-methylhexanoate could be converted to the corresponding aldehyde and then to the 6-carbon alcohol, (S)-3- methyl-1-pentanol, by an enzyme from the bacterium Lactococcus lactis (2-ketoisovalerate decarboxylase, KIVD) and another from the yeast Saccharomyces cerevisiae (alcohol dehydrogenase VI, ADH6).

They engineered three synthetic operons comprising 14 genes to overexpress all the enzymes. While the new pathway produced the 6-carbon sugar, the yield was low: one strain produced 6.5±1.1 mg/L of the 6-carbon alcohol from 20 g/L of glucose. Another strain produced 40.8±5.5 mg/L.

To improve yield, Liao and his team re-engineered two of the enzymes involved in the process. To reduce the formation of byproducts and drive the carbon flux toward the target C6 alcohol, they engineered KIVD with higher selectivity toward 2-keto-4-methylhexanoate. This resulted in production from one strain of 384.3±30.3 mg/L. They then modified LeuA, the other key enzyme determining the carbon flux toward 3-methyl-1-pentanol. This, combined with the KIVD mutant, pushed yield from one of the strains up to 793.5±46.5 mg/L.

For practical applications, further metabolic engineering and enzyme engineering will be needed to increase the production yield and rate of these compounds.

—Zhang et al. 2008

The work was supported in part by the UCLA Department of Energy Institute for Genomics and Proteomics.

Resources

  • Kechun Zhang, Michael R. Saway, David S. Eisenberg, and James C. Liao (2008) Expanding metabolism for biosynthesis of nonnatural alcohols. PNAS doi: 10.1073/pnas.0807157106

Comments

ejj

I wonder if the people in the European countries are going to riot in the streets over this....after all, like genetically modified food crops, this could unleash new strains of viruses and plagues upon the earth! ...ejj...

zard

why would they riot over this??? it's not going into their mouth...it's going into their CARS..

ejj

Zard: All it takes is a little GM product to get into the environment...there could be sheer global devastation! We need all the Europeans to riot and demonstrate in the streets now just like they did with GM crops! AAHHHHHH! This is sooooo scary! ...ejj...

Alex Kovnat

If the UCLA-developed process for producing C5 and C6 alcohols proves practical, we should test said compounds for suitability for spark-ignition and compression-ignition engines, and also aviation turbine engines. BTW I am not ignoring possible uses of C4, C5, etc. alcohols as chemical feedstocks.

Because Diesel engines have particulate and NOx problems, I would be interested in whether C5 and C6 alcohols would result in lower emissions than BioDiesel (methyl esters of fatty acids) or Fischer-Tropsch Diesel fuels.

sulleny

Alex - very interesting questions. If these higher energy density alcohols C5, C6 could replace diesel and the toxic emissions - it would consolidate liquid biofuel types.

Another interesting area would be to see if these GMs, applied to algae, would yield higher outputs than the E. coli bacterium and yeasts. If algae that photosynthesizes ethyl alcohol, could be modified to make higher C5,C6 synthetics - the yield issue might improve.

But I also agree with ejj. A good Euro street riot is great for the media feast and it improves morale in a downturn economy.

zard

Alex...what they make is not GMed...They just use bacteria to make something that doesn't exist naturally...

zard

or maybe GMed...but i think this is controlled cycle that doesn't affect enviroment

Henry Gibson

There are enough tons of coal being dug and there are enough barrels of oil being got out of the ground so that the researchers can be paid to see if they can make bacteria produce long chain alcohols.

All dogs are said to be genetically modified wolves; just that they were modified by breeding for specific characteristics rather than shooting gold spheres coated with DNA into cells.

Nature is also genetically modifying organisms with the help of viruses; it does not need humans.

Propane instead of methane would be nice for bacteria to make. It can be transported as liquid at low pressures, but makes a gas when released. Butane is a little bit too liquid below freezing. The need for extra large tanks makes hydrogen too heavy. Perhaps Ammonia is the best carbon free fuel. We risk demise when driving any way so what if ammonia is somewhat antibiotic. Sodium and air and water make a good source of energy, and the resulting sodium hydroxide will pickup CO2.

Even if it comes from solar cells, every bit of energy comes from nuclear reactions. There was no oxygen to burn hydrogen before it was made in stars.

There is no renewable energy. Renewable energy is just a falsification invented to disguise expensive forms of energy that failed already, over two centuries ago, to supply the wants of human organisms.

Nature does not conserve energy. Less than one two thousand millionth (1/2000,000,000) of the solar rays meet up with the earth and a very few of these photons ever convert ADP to ATP and thence CO2 and H2O to H12C6O6 (sugar) and O2 (oxygen). Plants took over the earth by creating one of the most deadly chemicals O2 and destroyed most of the organisms that inhabited the earth up till that point in time.

There is no reason not to use uranium and thorium to produce energy for humans as it is already destroying itself. Most of the U235 that was on the earth when it was formed is gone already and is now lead. Thorium atoms wait about 2,000,000,000 years and then expell a helium nucleus and becomes lead in about 25 years. Uranium does not take so long to start but takes longer to get all the way to lead. It stops for about 2000 years to become radium, the most dangerous natural element to be found naturally on the face of the earth except except that there is so little. Oxygen and sunlight are statistically far more dangerous.

There is enough Uranium in storage already to supply all the energy used by civilization for at least the next two hundred years if used in Rubbia Reactors. Much of this is in stored "used" fuel rods. The two thousand tons of "used" fuel rods produced by US nuclear reactors every year have enough energy remaining to produce 40,000,000,000,000 KWH of heat. This is enough to supply all the electricity of the US for five years using Rubbia Reactors (Accelerator Driven Sytems), or the same level of power for 25 years. If converted to a metal the remaining uranium fuel would be a 15 foot cube. All of the used uranium from all of the US reactors for a year could be put in a single small high ceiling room.

If all of the dangerous actual products of fission were removed from the used fuel they would form a cube about six feet on a side. This is all of the material that actually has to be stored for a years worth of operation of all the US reactors.

It might have taken about 20,000 tons of mined uranium to produce 2000 tons of fuel in the enrichment process, and this means that there is another 250 years of energy available in stored depleted uranium of one years enrichment process.

There is a high enough percentage of uranium in sea water to make it worth while to extract if used in Rubbia Reactors, but it will be a century or much more before it is economic compared to mines. This does not even consider the three times more abundant thorium.

Most energy efforts and money should be spent on developing processes for producing fuel from nuclear heat. Initially this will be done by using nuclear reactors to produce all electricity and diverting gas and coal to replace oil. Heat pumps will be used for heating and even cooking.

Then will come hydrogen production and then the loop will be closed by combining recaptured CO2 with hydrogen to produce methane or methanol which can be converted to gasoline or used directly.

There may be an electrolysis process invented that will use CO2 and water to produce methane directly. Some CO2 can be converted to charcoal to be mixed with soil to enhance fertility, but before that will come the conversion of biomass to charcoal for the same purpose instead of putting the CO2 back into the air. ..HG..

Richard Burton

Henry; "there is no reason not to use nuclear...." I beg to disagree. As I understand it, the more spent fuel out there the more likely it is to be diverted, into the potential for a "dirty bomb", or centrifuged into weapons grade material. And apparently, if one is trained as a nuclear engineer to run a power plant, you have all the necessary skills to make a weapon.
I think this is a huge drawback! imagine a dirty bomb going off in the port of Los Angeles- it would contaminate and render several square miles of city uninhabitable for a very long time. Feel comfortable with the nuclear weapons and fuel that is in Pakistan right now? Richard

Mannstein

Actually contaminating Los Angeles has an upside. It would solve the gang problem.

arnold

Henry,
I enjoyed your story up to paragraph eight.
then I would like to take that paragraph eight out (of context) and analyse it.
After Reading this I am left wondering how concentrated and dangerous it appears, not that I can't see the point that you make as far as energy, resource potential etc, just that all this stuff has dangers assosiated that are a little more serious than being personally injured in a circumstance that offers many options for avoidance or persomnal choice.That we have with flying, motor vehicle use, alcohol tobacco or other behaviours.

In contrast the potential for damage is silent and unlikely to be related to ones actions.
If you were majicaly to hold the mantle 'Saviour' for the purposes of implementing these works, could you assure me that you wont make a mistake?
Arn.

"There is enough Uranium in storage already to supply all the energy used by civilization for at least the next two hundred years if used in Rubbia Reactors. Much of this is in stored "used" fuel rods. The two thousand tons of "used" fuel rods produced by US nuclear reactors every year have enough energy remaining to produce 40,000,000,000,000 KWH of heat. This is enough to supply all the electricity of the US for five years using Rubbia Reactors (Accelerator Driven Sytems), or the same level of power for 25 years. If converted to a metal the remaining uranium fuel would be a 15 foot cube. All of the used uranium from all of the US reactors for a year could be put in a single small high ceiling room."

Thats one mighty powerful concoction.

Any one who passes a college level physics class has the skill set to make an atomic weapon provided the materials. The basic physics for a device was solved in the 1940's with sliderules and charts. even a boosted fusion fission device is with in the grasp of most college level engineering students these days. On access to the materials needed keep the dragon at bay, hence the reason to stop Iran from having enrichment tech once they have enough U235 its a matter of 1940 tech to bring 2 subcritial masses together at >200 Meters per second relative velocity.

Mark_BC

I think the "bad guys" already have the bombs.

jimbo

Nuclear presents a problem worse than all the those associated with its lethality. Because of the technical issues of mining, refining and handling it can be manipulated by those who will use it to control populations, just as is happening with oil. Nuclear is in a big sweat now because if "renewable" (if I'm allowed to use that term) resourses continue to be developed then there will be thousands of sources of energy, all in competition with one another, employing millions of people. All of the energy will be clean, there will never again be an issue about poisoning of mankind, whether it is CO2 or radiation. So Henry, put it to bed, the nuclear issue is over.

danm

henry makes the very valid point that it's unlikely "renewable" energy can satisfy our demand (unless, perhaps, we have vast algal farms floating on the oceans some day?)
Unfortunately, he simplifies all the problems with nuclear and solves them with nonexistant technology. For example, Rubbia reactors and reducing nuclear waste to a 6 ft cube(great idea but vastly expensive).

Nuclear still holds great promise, but only if its advocates face the problems honestly. (same can be said for renewables and fossils).

Mark_BC

danm, you should do a calculation of the potential of solar power. If you do, you will find that to power the entire US, it would require about 600 km by 600 km of solar panels in the southwest states. This is not very much, considering that: 1) commercial PV efficiency will likely double in the next few years, 2) capable fully electric cars will be available in the next few years, 3) the rest of the country can also produce its own solar electricity, though not as much as the desert southwest, 4) it is expected that solar electricity will reach grid parity in California without subsidies in about 5 years, 5) most of that required surface area could be found on rooftops throughout the region which are otherwise wasted space.

In less than 20 years there will be no more energy crisis.

reel$$

Not to mention that there is an entirely unproductive and unnecessary lid on Low Energy Nuclear Reactions - for which there is now more than twenty years of laboratory proof.

Or the work that Randy Mills is doing with atomic H and "hydrinos" over at BlackLight Power.

Or the various overunity experiments using cavitated plain water.

Or rare earth magnetic motors...

There is only an energy "problem" for one universal reason: certain people profit from it.

danm

Marc_BC, i honestly hope you're optimism is correct. my doubts stem from facts such as: all solar panels manufactured to date equal less than a sq km. ...can we capture enough power for a car? (granted, it would be a much different car that we're used to driving) and capture enough for all manufacturing? trucking? RR's

Mannstein

Marc_BC, you forgot to mention what solar arrays in the desert Southwest will do to the California ground squirrel.

Isn't it clear by now that for every technical solution to the energy crisis there are at least 100 green jerks trying to shoot it down.

I'm heating with coal and loving it.

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