Valine and alpha-KIV can be transported out to the cytoplasm, where they are converted by a set of enzymes into isobutanol. Other researchers have tried to express all the enzymes needed for isobutanol biosynthesis in the cytoplasm. However, it's difficult to get some of those enzymes to function in the cytoplasm as well as they do in the mitochondria.
The MIT researchers took the opposite approach: They moved the second phase, which naturally occurs in the cytoplasm, into the mitochondria. They achieved this by engineering the metabolic pathway's enzymes to express a tag normally found on a mitochondrial protein, directing the cell to send them into the mitochondria.
This enzyme relocation boosted the production of isobutanol by 260 percent, and yields of two related alcohols, isopentanol and 2-methyl-1-butanol, went up even more 370 and 500 percent, respectively.
There are likely several explanations for the dramatic increase, the researchers say. One strong possibility, though difficult to prove experimentally, is that clustering the enzymes together makes it more likely that the reactions will occur, Avalos says.
Another possible explanation is that moving the second half of the pathway into the mitochondria makes it easier for the enzymes to snatch up the limited supply of precursors before they can enter another metabolic pathway.
"Enzymes from the second phase, which are naturally ou
|Contact: Caroline McCall|
Massachusetts Institute of Technology