C. Dellomonaco et al. pieced together decades of research in bacterial genetics and biochemistry to produce a tour de force of genetic engineering, John Timmer of Ars Technica reports:
Cells have a pathway for the production of fatty acids, long hydrocarbon chains that are normally linked together to form fats. The end products at least look a bit more like the fuels that currently run our cars than ethanol does, but using this pathway to produce biofeuls has drawbacks. It requires a substantial input of energy in the form of ATP and tends to produce hydrocarbon chains that are too long (10-20 carbons long) to make a really convenient fuel. This pathway is also tightly regulated, since most microbes would rather divert their energy to reproduction than to making fat.
As a result, a team of researchers from Rice University decided to forgo this pathway entirely. They reasoned that cells have a second, entirely separate set of enzymes normally used to break fats down that might be repurposed to make biofuel.
Enzymes are catalysts. They generally act by making a chemical reaction more likely to occur — they don’t usually dictate in which direction the reaction goes. So, if you supply an enzyme with a large quantity of what are normally the end products of a given reaction, it will readily catalyze the reverse reaction. If you run the pathway that normally digests fats in reverse, it will produce longer hydrocarbons.
Sounds simple, right? But actually getting bacteria (the authors worked with E. coli) to do this isn’t necessarily easy. To begin with, the bacteria won’t produce any of these necessary enzymes unless they think they have fat to digest. Years of genetic studies have identified the genes responsible for shutting off the fat burning pathway, so the authors knocked those genes out.
Problem solved? Not quite. Even when fat is available, E. coli would rather burn simple sugars instead if they’re present. The gene that mediates this preference has also been identified, and the authors spliced a mutant form of it into the bacteria’s DNA. With these mutations in place, the bacteria would at last have the right enzymes around, no matter what the conditions.
The authors fed their modified E. coli glucose, which can be produced by the breakdown of cellulose (meaning the process is biofuel compatible). Glucose is a six-carbon molecule that’s broken down into short, two-carbon chunks in a process that produces ATP to fuel the cell. These two carbon molecules end up attached to a co-factor in a molecule called acetyl-Coenzyme A. If oxygen is present, acetyl-CoA gets handed over to a process that produces a number of ATP molecules as acetyl-CoA is converted into water and carbon dioxide (the CoA is recycled). If oxygen is not present, organisms like yeast convert acetyl-CoA into ethanol instead, freeing up the CoA for reuse.
As it turns out, acetyl-CoA is also where the digestion of fats feeds into the normal metabolism. So, by giving the bacteria lots of glucose, the authors created conditions where the end product of fat digestion, acetyl-CoA, was present in abundance, but there wasn’t an excess of the starting material, namely fat. This was enough to tip the pathway backwards, building up longer chains of hydrocarbons. To give the system an extra boost, the authors knocked out the gene that sends acetyl-CoA down the pathway towards ethanol.
On its own, this process wouldn’t do anything useful, since it would create a mix of longer hydrocarbons all linked up to coenzyme A. But organisms have ways of diverting specific products for use in the production of specific molecules they need, such as amino acids or the bases of DNA. So the authors did a bit more engineering and added some copies of the gene that divert a four-carbon intermediate into butanol. Expression of a different gene shifted the production toward longer hydrocarbons, resulting in a mix of molecules that contain a chain of 12 to 18 carbon atoms. Almost all of the reactions researchers tested resulted in the most efficient production of end products that anyone has reported.
Peak what now?