Researchers at MIT have modified the soil bacterium Ralstonia eutropha to produce isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. The work is funded by the US Department of Energy’s Advanced Research Projects Agency – Energy (ARPA-E). (Earlier post.) A paper on their progress is published in the journal Applied Microbiology And Biotechnology.
When under nutrient stress and in the presence of excess carbon, wild-type Ralstonia eutropha H16 stops growing and begins producing polyhydroxybutyrate (PHB)—a complex carbon compound—as an intracellular carbon storage material during nutrient stress in the presence of excess carbon.
What it does is take whatever carbon is available, and stores it in the form of a polymer, which is similar in its properties to a lot of petroleum-based plastics.—Christopher Brigham, co-author of the paper
The MIT team redirected the carbon in the engineered strains from PHB storage to the production of the alcohols. They used various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, for the biosynthesis of new products.
Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered bacteria.
One mutant strain not only produced more than 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three other potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol.
Christopher Brigham, co-author of the paper, has been trying to get the organism to use a stream of carbon dioxide as its source of carbon, so that it could be used to make fuel out of emissions.
While the team is focusing on getting the microbe to use CO2 as a carbon source, with slightly different modifications the same microbe could also potentially turn almost any source of carbon, including agricultural waste or municipal waste, into useful fuel. In the laboratory, the microbes have been using fructose, a sugar, as their carbon source.
At this point, the MIT team—which includes chemistry graduate student Jingnan Lu, biology postdoc Claudia Gai, and is led by Anthony Sinskey, professor of biology—have demonstrated success in modifying the microbe’s genes so that it converts carbon into isobutanol in an ongoing process.
Now, the researchers are focusing on finding ways to optimize the system to increase the rate of production and to design bioreactors to scale the process up to industrial levels.
Unlike some bioengineered systems in which microbes produce a wanted chemical within their bodies but have to be destroyed to retrieve the product, R. eutropha naturally expels the isobutanol into the surrounding fluid, where it can be continuously filtered out without stopping the production process, Brigham says.
We didn’t have to add a transport system to get it out of the cell.—Christopher Brigham
Jingnan Lu, Christopher J. Brigham, Claudia S. Gai and Anthony J. Sinskey (2012) Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha. Applied Microbiology And Biotechnology doi: 10.1007/s00253-012-4320-9