Is isobutanol the petroleum of tomorrow?
20 March 2013
Heavy alcohols such as isobutanol are showing real potential in researchers’ efforts to find a renewable alternative to petroleum. Firstly, they contain more energy than ethanol, but they are also more compatible with existing petroleum-based infrastructure. However, isobutanol production must be reliable and large-scale for its use to be considered truly practical. It must also be available from renewable resources.
It has long been known that yeast makes isobutanol naturally, albeit in small amounts. However, chemical engineers and biologists from Massachusetts Institute of Technology (MIT) have discovered a method of boosting isobutanol production in yeast by some 260 per cent, by engineering the yeast so that the synthesis of isobutanol takes place entirely within mitochondria.
Although this would still fail to meet the economies of scale necessary for industrial production, experts believe that it is an important advance in the engineering of not only isobutanol, but also other chemicals. Gregory Stephanopoulos is an MIT professor of chemical engineering and one of the senior authors of a paper describing the work in the February 17 online edition of Nature Biotechnology.
“It’s not specific to isobutanol,” according to Prof Stephanopoulos. “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.”
This new push to encourage isobutanol production in yeast runs counter to the traditional approach that has been taken until now, whereby researchers have tried to prevent the development of isobutanol – as it can ruin the flavour of beer and wine.
During fermentation, yeast cells can make small amount of isobutanol, an alcohol that contains more energy than ethanol and is more suitable as a transport biofuel. The natural production process for isobutanol in yeast consists of stages that are compartmentalised in cell mitochondria and cytoplasm.
Yeast typically produces 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 is difficult to get some of those enzymes to function in the cytoplasm as well as they do in the mitochondria.
The researchers from MIT instead moved the second phase into the mitochondria, rather than the cytoplasm, where it occurs naturally. In order to do this, they engineered the enzymes of the metabolic pathway to express a tag normally found on a mitochondrial protein, directing the cell to send them into the mitochondria.
The relocation of this enzyme had the effect of boosting the production of isobutanol by 260 per cent. In addition, it also increased the yields of two other alcohols – isopentanol and 2-methyl-1-butanol – by 370 per cent and 500 per cent, respectively.
According to the researchers, the dramatic increase may be due to a number of factors: it was most likely that clustering the enzymes together made it more likely that the reactions would occur. However, this theory was difficult to prove experimentally, they added.
There may also be another explanation: by moving the second half of the pathway from the cytoplasm into the mitochondria, the enzymes were better able to maximise the limited supply of precursors before they entered 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,” explained Jose Avalos, a postdoctoral student at MIT and the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts.
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.
Currently, the researchers are working to further boost isobutanol yields and reduce production of ethanol, which is still the major product of sugar breakdown in yeast.
“Knocking out the ethanol pathway is an important step in making this yeast suitable for production of isobutanol,” Prof Stephanopoulos said. “Then we need to introduce isobutanol synthesis, replacing one with the other, to maintain everything balanced within the cell.”
Thus far, the MIT researchers have persuaded many that the idea of using yeast to produce large yields of isobutanol is a viable option that merits further exploration. The next step is to see if maximising the process can produce enough isobutanol to exploit commercially.
The research was funded by the National Institutes of Health and Shell Global Solutions. This article is based on a release issued by the MIT News Room.