MODIFIED yeast that use light as a switch to change metabolic pathways have been used to produce the biofuel isobutanol.
Metabolism is the basic chemical process in living cells and it can be manipulated to perform different functions such as the production of valuable chemicals, fuels, or drugs. Fermentation is a metabolic process and one example is the transformation of sugars to ethanol with yeast. Pyruvate is produced from sugar during one of the fermentation steps, and the preferred metabolic pathway for yeast is to catalyse the pyruvate with the enzyme pyruvate decarboxylase to produce ethanol.
Natural yeast fermentation also produces miniscule amounts of isobutanol, an alcohol that can be used as a substitute for petrol, and also used in products such as lubricants and plastics. Researchers at the US’ Princeton University have devised a method to get yeast to produce substantially more isobutanol – an impressive feat as high doses of isobutanol will kill yeast.
They genetically modified the yeast to include a light-sensitive gene from a marine bacterium. This leaves the yeast susceptible to optogenetics – the manipulation of genes via light. By exposing the yeast to blue light, they can switch different metabolic pathways on or off.
"This technique allows us to control the metabolism of cells in an unprecedented way," said co-lead researcher José Avalos, assistant professor of chemical and biological engineering. "It provides a new tool with the ability to do sophisticated experiments to determine how metabolism works and how to engineer it.”
When the yeast is exposed to light it undergoes a growth phase and produces ethanol as normal. A darkness-induced production phase can be used to produce either lactate, which is used in food production and bioplastics, or isobutanol. Before the yeast die from the isobutanol exposure, the light can be switched back on to stimulate another growth phase.
"Normally light turns expression on but we also had to figure out how to make the absence of light turn another expression on," said Jared Toettcher, assistant professor of molecular biology and co-lead researcher. “Just enough light to keep the cells alive but still crank out a whole lot of product that you want, which they produce only in the dark.”
The production phase changes the metabolic pathway of the pyruvate so that biosynthesis of alternative pyruvate-derived products can occur. Distinct enzymes can act on the pyruvate in what are ordinarily competing pathways. In the case of lactate, the enzyme is lactate dehydrogenase, and acetolactate synthase is one of the enzymes used for isobutanol production.
This is the first time that optogenetics has been used to produce chemicals. Previous work to produce isobutanol from yeast used alternative methods involving genetically deleting pathways that competed with the desired pathway. The light-controlled method increased the production of isobutanol five-fold compared to previous work. Light is also much faster and cheaper than chemical alternatives and it can easily by switched on and off so that it can be applied to specific genes without affecting other parts of the cell.
Future work will look at using different colours of light to activate different proteins. “We intend to keep pushing,” said Avalos. “But metabolic engineering transcends industrial microbiology. It also allows us to study the metabolism of cells for health-related problems. You can control metabolism in any context, for industrial biology or to address medical questions.”
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