MIT team shows targeting metabolic pathways to mitochondria significantly boosts yeast production of isobutanol; potential for other chemicals as well
Researchers from MIT and the Whitehead Institute for Biomedical Research have devised a way to boost significantly isobutanol production in yeast by engineering isobutanol synthesis to take place entirely within mitochondria.
They showed that targeting metabolic pathways to mitochondria can increase production compared with overexpression of the enzymes involved in the same pathways in the cytoplasm. Compartmentalization of the Ehrlich pathway—a three-step catalytic breakdown of valine that produces isobutanol, earlier post—into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overproducing enzymes involved in only the first three steps of the biosynthetic pathway. A paper on their work is published in the journal Nature Biochemistry.
Metabolic engineering of cytoplasmic biosynthetic pathways to create industrial strains of S. cerevisiae is commonplace, whereas engineering of biosynthetic pathways that function in mitochondria has largely been ignored. Yet mitochondria have many potential advantages for metabolic engineering, including the sequestration of diverse metabolites, such as heme, tetrahydrofolate, ubiquinone, α-ketoacids, steroids, aminolevulinic acid, biotin and lipoic acid. In addition, mitochondria contain intermediates of many central metabolic pathways, including the tricarboxylic acid (TCA) cycle, amino-acid biosynthesis and fatty-acid metabolism.
The environment in the mitochondrial matrix differs from that in the cytoplasm: it has higher pH, lower oxygen concentration and a more reducing redox potential. This environment may more closely match the optimal for maximal activity of many enzymes such as the iron-sulfur clusters (ISCs), which are essential cofactors of enzymes in diverse pathways including branched-chain amino acid and isoprenoid biosynthetic pathways, and which are synthesized exclusively in mitochondria. Although ISCs can be exported to the cytoplasm, the molecular machinery that loads ISCs onto extramitochondrial enzymes is likely to be incompatible with most exogenous ISC apoenzymes, especially those of bacterial or archaeal origin. The smaller volume of mitochondria could concentrate substrates favoring faster reaction rates and productivity, and confine metabolic intermediates, avoiding repressive regulatory responses, diversion of intermediates into competing pathways or even toxic effects of intermediates to cytoplasmic or nuclear processes.—Avalos et al.
Though still short of the scale needed for industrial production, the advance suggests that this is a promising approach to engineering not only isobutanol but other useful chemicals as well, says Gregory Stephanopoulos, an MIT professor of chemical engineering and one of the senior authors of the paper.
It’s not specific to isobutanol. It’s opening up the opportunity to make a lot of biochemicals inside an organelle that may be much better suited for this purpose compared to the cytosol of the yeast cells.—Gregory Stephanopoulos
Stephanopoulos collaborated with Gerald Fink, an MIT professor of biology and member of the Whitehead Institute, on this research. The lead author of the paper is José Avalos, a postdoc at the Whitehead Institute and MIT.
Yeast typically produce isobutanol in a series of reactions that take place in two different cell locations. The synthesis begins with pyruvate, a plentiful molecule generated by the breakdown of sugars such as glucose. Pyruvate is transported into the mitochondria, where it can enter many different metabolic pathways, including one that results in production of valine, an amino acid. Alpha-ketoisovalerate (alpha-KIV), a precursor in the valine and isobutanol biosynthetic pathways, is made in the mitochondria in the first phase of isobutanol production.
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%, and yields of two related alcohols, isopentanol and 2-methyl-1-butanol, went up even more—370% and 500%, respectively.
There are likely several explanations for the 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 out here in the cytoplasm, have to wait to see what comes out of the mitochondria and try to transform that. But when you bring them into the mitochondria, they’re better at competing with the pathways in there.—José Avalos
The findings could have many applications in metabolic engineering. There are many situations where it could be advantageous to confine all of the steps of a reaction in a small space, which may not only boost efficiency but also prevent harmful intermediates from drifting away and damaging the cell.
The researchers are now trying to further boost isobutanol yields and reduce production of ethanol, which is still the major product of sugar breakdown in yeast.
The research was funded by the National Institutes of Health and Shell Global Solutions.
José L Avalos, Gerald R Fink & Gregory Stephanopoulos (2013) Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nature Biotechnology (2013) doi: 10.1038/nbt.2509