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Thinking Inside the Box (or Mitochondrion)

March 28, 2013

The mantra in real estate is that location is everything. The same may be true for some cases of bioengineering. You may not get the best yield unless a whole pathway is in the right cellular compartment.

cat in a box

Sometimes it helps to think inside the box.

This is what Avalos and coworkers found for synthesizing branched chain alcohols in the yeast Saccharomyces cerevisiae. These scientists were able to increase yield by 260% by putting the whole pathway of enzymes into the mitochondrion. This is way better than anything anyone else has been able to achieve.

This matters because branched alcohols like isobutanol may prove to be better biofuels than ethanol. We can get more energy out of isobutanol than we can out of ethanol…it has more bang for the buck. Good idea in theory, but producing large quantities of isobutanol has not worked too well in practice.

Yeast is just not very good at making these alcohols, and efforts to improve yields have been anything but inspiring so far. Overexpressing the enzymes in the metabolic pathways that generate isobutanol increased yield by only about 10%. Unfortunately, 10% of almost nothing is still pretty close to nothing.

One of the key metabolic pathways involved in generating isobutanol and other branched chain alcohols is split between the mitochondria and the cytoplasm. Normally, the valine biosynthesis pathway converts pyruvate to valine and alpha-ketoisovalerate in the mitochondria; then those two intermediates, after transport to the cytoplasm, are further converted to isobutanol by the Erlich pathway for valine degradation. Avalos and coworkers reasoned that the failure to increase yield might be because of some rate limiting step in getting the intermediates from the mitochondria to the cytoplasm. And it looks like they may have been right.

They compared the effects of overexpressing the pathway enzymes in the cytoplasm and mitochondria and found the mitochondrial approach won hands down. Overexpression in the cytoplasm bumped yield up 10% while overexpression bumped it up 260%. And this increase wasn’t just for isobutanol. Yields of two other energy rich alcohols, isopentanol and 2-methyl-1-butanol, also went up significantly.

Part of the explanation almost certainly has to do with transport of intermediates between the mitochondrion and the cytoplasm, but that may not be the whole story. The mitochondrion might be a useful environment for other reasons too. For example, its smaller volume means an increase in the concentration of reactants, and its higher pH, lower oxygen content, and more reducing redox potential may be better for certain reactions. It also contains many key intermediates like heme, steroids, biotin and so on.

On the way to improving isobutanol yield, these scientists made it easier for others to test whether moving their pathway to the mitochondria can help increase the yield of their favorite metabolite. Avalos and coworkers created a system of plasmids that easily allows researchers to attach the N-terminal mitochondrial localization signal from Cox4p, subunit IV of the yeast cytochrome c oxidase, to genes of their choice. This will make it much simpler to test whether a pathway’s yield is enhanced by moving it into the mitochondrion.

These results show there is more to increasing yield than overexpression or codon optimization. Sometimes scientists need to take a good hard look at their particular pathway and think outside of the box for new ways to optimize yield. Or sometimes they just need to think within the box that is the mitochondrion.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics