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UC Berkeley researchers boost E. Coli butanol production with new enzymatic pathway

Researchers at the University of California, Berkeley, have constructed a chimeric pathway assembled from three different organisms for the high-level production of n-butanol (4,650 ± 720 mg l-1) in E. coli bacteria. The pathway uses an enzymatic chemical reaction mechanism in place of a physical step as a kinetic control element to achieve high yields from glucose (28%).

The advance is reported in this week’s issue of the journal Nature Chemical Biology by Michelle C. Y. Chang, assistant professor of chemistry at UC Berkeley, graduate student Brooks B. Bond-Watts and recent UC Berkeley graduate Robert J. Bellerose.

Various species of the Clostridium bacteria naturally produce n-butanol using five enzymes that convert a common molecule, acetyl-CoA, into n-butanol. Other researchers, including a few biofuel companies, have genetically altered Clostridium to boost its ability to produce n-butanol, while others have plucked enzymes from the bacteria and inserted them into other microbes, such as yeast, to turn them into n-butanol factories. Yeast and E. coli, one of the main bacteria in the human gut, are considered to be easier to grow on an industrial scale.

However, n-butanol is not produced rapidly in these systems because the native enzymes can work in reverse to convert butanol back into its starting precursors. While these techniques have produced promising genetically altered E. coli bacteria and yeast, n-butanol production has been limited to little more than half a gram per liter, far below the amounts needed for affordable production.

Chang and her colleagues placed the same enzyme pathway into E. coli, but replaced two of the five enzymes with look-alikes from other organisms that avoided one of the problems other researchers have had: n-butanol being converted back into its chemical precursors by the same enzymes that produce it.

Chang avoided this problem by searching for organisms that have similar enzymes, but that work so slowly in reverse that little n-butanol is lost through a backward reaction.

Depending on the specific way an enzyme catalyzes a reaction, you can force it in the forward direction by reducing the speed at which the back reaction occurs. If the back reaction is slow enough, then the transformation becomes effectively irreversible, allowing us to accumulate more of the final product.

—Michelle Chang

Chang found two new enzyme versions in published sequences of microbial genomes, and based on her understanding of the enzyme pathway, substituted the new versions at critical points that would not interfere with the hundreds of other chemical reactions going on in a living E. Coli cell. In all, she installed genes from three separate organisms—Clostridium acetobutylicum, Treponema denticola and Ralstonia eutrophus—into the E. coli.

The new genetically altered E. Coli produced nearly five grams of n-butanol per liter, about the same as the native Clostridium and one-third the production of the best genetically altered Clostridium, but about 10 times better than current industrial microbe systems.

We are in a host that is easier to work with, and we have a chance to make it even better. We are reaching yields where, if we could make two to three times more, we could probably start to think about designing an industrial process around it.

—Michelle Chang

Chang is also at work adapting the new synthetic pathway to work in yeast.

The work was supported by UC Berkeley, the Camille and Henry Dreyfus Foundation, the Arnold and Mabel Beckman Foundation and the Dow Sustainable Products and Solutions Program.


  • Brooks B Bond-Watts, Robert J Bellerose, Michelle C Y Chang (2011) Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. Nature Chemical Biology doi: 10.1038/nchembio.537



Good news for future cellulosic butanol production.

Henry Gibson

However good bio-butanol is produced there is not enough bio-mass to produce it in sufficient quantities.

Humans forget that they produce a lot of CO2 by breathing and far more with their mechanical monsters. Radioactive potassium is in every human, animal and plant and allows the voltages for nerve operation to be generated. This nerve operation has allowed the creation of monsters that spew CO2 into the air, but it also permits false beliefs that hinder the use of the cheapest lowest CO2 energy production.

The main false belief is that humans can be given perfect safety by eliminating dangerous things and substances whilst they are choosing to use the statistically most dangerous combinations of technology ever invented by humans especially cell phones, ethanol and automobiles.

Not another penny should be spent on reducing the danger of radioactive materials thousands of years in the future until the deaths of people, now because of the operation of texting in automobiles, is eliminated.


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