E. coli Takes On Biofuels

Petroleum constitutes the main source of energy all over the world. Excessive consumption of this resource due to the continuous increase in the world’s population has led to the depletion of petroleum reserves, which coincides with the increasing impacts of climate change due to the release of greenhouse gas emissions (Stephanopoulos, 2007).

A potential solution to these problems lies in biofuels, alternative sources of sustainable energy that can be generated from plants such as corn, Poplar, and switchgrass. 

The first major alternative biofuel used in engines was bioethanol, commonly known as ethanol, in the early 1990s, which was blended with gasoline to create 10% ethanol and 90% gasoline E10 fuel. Other blends include E15, which includes up to 15% ethanol, and E85, which can be used in flexible fuel vehicles. Today, more than 98% of the fuel used in the United States is E10 blended fuel, making this a common and convenient fuel alternative.

However, this is not the most efficient renewable fuel available. While ethanol has provided a solid starting point for biofuel use precedence, this particular biofuel has inherent challenges which limit further expansion into the fuel economy, including a low amount of stored energy and corrosive effects which lead to cracks in the steel used to manufacture engine parts (Clemente, 2015). 

As a result, researchers have looked to optimize more efficient alternative biofuels using genetic engineering in the common bacteria Escherichia coli. For most of us, E. coli is only mentioned in food contamination news reports. In the genetic engineering world, E. coli is a valuable user-friendly host with a well-known growth metabolism and the largest set of genetic engineering tools available. Many people may be surprised to know that this bacteria has been studied extensively for gene regulation and expression, and has served as a source of valuable substances for human use, such as insulin production for diabetes treatment. Escherichia coli is able to consume various sugars, allowing for versatile growth conditions at both laboratory and industrial scales, making E. coli the organism of choice for genetic engineering. 

The advancement of genetic engineering has enabled geneticists to modify genes to produce more or less of a specific potential biofuel, such as isopropanol. These modifications alter the genetic makeup of the organism by introducing a new gene or modifying an existing one. This can be done by changing a DNA base pair (adenine-thymine/guanine-cytosine), deleting a whole gene, or inserting multiple genes. Tools which allow biological engineers to transfer entire metabolic pathways from one organism to another have been developed, allowing scientists to work with more easily engineered organisms in a laboratory. These tools are generally available for use in organisms such as viruses and bacteria, and transferring a gene or sequence of genes into a different organism allows this organism to produce a molecule of interest, such as isopropanol. 

Genetic engineering process. Image courtesy of Angel Fernando Cisneros Caballero

Acetone and isopropanol are sustainable, cheap, have high energy densities, and have been tested as alternative fuels. These biofuels are already compatible with existing engines (Poh & Poh, 2017), and isopropanol has a higher blending capability than ethanol with lessened corrosive effects. Just like isopropanol, acetone has a higher energy density and blending capacity in comparison to ethanol and is also less corrosive to engines. All of these traits mean that both isopropanol and acetone have the potential to complement or replace ethanol in the biofuel market, and both of these are naturally produced by a bacterium called Clostridium acetobutylicum. 

Clostridium acetobutylicum produces isopropanol and acetone via a single metabolic pathway, meaning that this organism produces both of these potential biofuels using the same sequence of genes. Acetone is produced as the primary product of this gene pathway, and isopropanol is obtained from acetone as a by-product. The pathway is not designed to maximize the production of these two molecules, but it can be engineered to do so. Unfortunately, genetic engineering tools which can be used with C. acetobutylicum are limited, and several other biochemical pathways must be inhibited to optimize the biofuel yields (Toogood & Scrutton, 2018).

Fortunately, the acetone pathway of C. acetobutylicum can be transferred into E. coli, leading to the production of acetone and isopropanol without the limitations of C. acetobutylicum. Genetic engineers can “over-express” the genes used to produce acetone and isopropanol in E. coli, meaning that these genes can be pushed into overdrive to produce much higher amounts of isopropanol and acetone for potential biofuel use (Atsumi & Liao, 2008; Koppolu & Vasigala, 2016). 

Advances in genetic engineering have enabled scientists to study and improve an organism’s natural metabolic pathways to produce compounds which humans can then mass produce for health, energy, and food benefits. The ability to transfer an entire metabolic pathway from C. acetobutylicum into E. coli may lead to commercially viable applications of an organism’s natural abilities to produce efficient, high-quality biofuels that can improve or replace traditional fuel, thereby alleviating emerging supply and environmental problems associated with ethanol. Challenges still remain with engine and fuel mixture optimizations to reduce emissions and improve the energy yield, but current results are encouraging in our mission to reduce our dependence on petroleum.

These findings are based on work conducted by Jessica Velez from the Bredesen Center for Interdisciplinary Studies at the University of Tennessee, Knoxville, Angel Fernando Cisneros Caballero from the Institute of Integrative and Systems Biology at Laval University, and Narjes Alfuraiji from the Division of Immunity, Infection and Respiratory Medicine at the University of Manchester.

References: 

1. Stephanopoulos G., (2007). Challenges in engineering microbes for biofuels production, Science, 315(5813), 801–804. 
2. Clemente, J. Why biofuels can’t replace oil. Forbes. 2015. https://www.forbes.com/sites/judeclemente/2015/06/17/why-biofuels-cant-replace-oil/#467b62f6f60f 
3. Poh, C., & Poh, C., (2017). Isopropanol as fuel for small unmanned aircraft, Advances in Aerospace Science and Technology, 2, 23-30.  
4. Atsumi, S., & Liao, J., (2008). Metabolic engineering for advanced biofuels production from Escherichia coli, Current Opinion in Biotechnology
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6. Koppolu, V., & Vasigala, V., (2016). Role of Escherichia coli in biofuel production, Microbiology Insights, 9, 29-35.
7. Toogood, H., & Scrutton, N., (2018). Retooling microorganisms for the fermentative production of alcohols, Current Opinion in Biotechnology, 50, 1-10.