In a breakthrough that defies nature, Northwestern University and Stanford University synthetic biologists have created a new artificial metabolism that transforms waste carbon dioxide (CO2) into useful biological building blocks.
In the new study, the team engineered a biological system that can convert formate — a simple liquid molecule easily made from CO2 — into acetyl-CoA, a universal metabolite used by all living cells. As a proof of concept, the engineers then used the same system to convert acetyl-CoA into malate, a commercially valuable chemical used in foods, cosmetics and biodegradable plastics.
Unlike natural metabolic routes, the new system is entirely synthetic and operates outside of living cells. The engineers built the system, called the Reductive Formate Pathway (ReForm), from engineered enzymes that performed metabolic reactions never before seen in nature.
The work marks a major advance for synthetic biology and carbon recycling, opening the door for developing sustainable, carbon-neutral fuels and materials.
The study will be published on Monday (Dec. 22) in the journal Nature Chemical Engineering.
“The unabated release of CO2 has caused many pressing social and economic challenges for humanity,” said Northwestern’s Ashty Karim, who co-led the study. “If we’re going to address this global challenge, we critically need new routes to carbon-negative manufacturing of goods. While nature has evolved several pathways to metabolize CO2, it is unable to keep up with the rapid increase in the amount of atmospheric CO2. Inspired by nature, we sought to use biological enzymes to convert formate derived from CO2 into more valuable materials. Because there isn’t a set of enzymes in nature that can do that, we decided to engineer one.”
“ReForm can readily use diverse carbon sources, including formate, formaldehyde and methanol,” said Stanford’s Michael Jewett, who co-led the study with Karim. “This is the first demonstration of a synthetic metabolic pathway architecture that can do so. By combining electrochemistry and synthetic biology, the ReForm pathway also expands possible solutions for generalizable CO2-fixation strategies. We anticipate that hybrid technologies that integrate the best of chemistry and the best of biology will provide transformative new directions for a carbon- and energy-efficient future.”
An expert in synthetic biology and biotechnology, Karim is an assistant professor of chemical and biological engineering at Northwestern’s McCormick School of Engineering and a member of the Center for Synthetic Biology (CSB). Jewett is an adjunct professor at Northwestern, founding co-director of CSB and a professor of bioengineering at Stanford.
Looking beyond nature
As researchers search for solutions to help fight the ever-warming atmosphere, many have sought to upcycle captured CO2 into valuable chemicals. Because it’s easy to make from electricity and water, formate has emerged as a promising starting point. Then, biological systems could perform the work needed to convert formate into useful materials.
But, unfortunately, living cells struggle to use formate efficiently. Only a few rare microbes can digest formate naturally, and those microbes are difficult to engineer for large-scale production.
“Cells naturally use metabolic reactions to convert one chemical into another,” Karim said. “For example, cells can take glucose, or sugar, and convert it into energy. But, in nature, nothing can turn formate into acetyl-CoA. There are some enzymes that can act on formate, but they cannot build it up into something useful. So, we started with a theoretical pathway design and the need for enzymes with functionalities that did not exist in nature.”
Testing thousands of enzymes per week
Before building the metabolic pathway, the research team needed enzymes that could perform these non-natural reactions. To rapidly express and test large numbers of enzyme variants, the team turned to cell-free synthetic biology. In this approach, scientists essentially remove a cell’s wall, collect its molecular machinery (enzymes, cofactors and small molecules) and put it all into a test tube. Scientists then can use this machinery — outside of a living organism — to make a product in a safe, inexpensive and rapid manner.
“It’s like opening the hood of a car and removing the engine,” Jewett said. “Then, we can use that ‘engine’ for different purposes, free from the constraints of the car.”
Using a cell-free system enabled the team to rapidly screen 66 enzymes and more than 3,000 enzyme variants to find the ones that worked best. This process was much faster and more flexible than using live cells, which would have been slow and laborious.
“Typically, people will test a handful of enzymes, and that takes months or more,” Karim said. “The cell-free environment enabled us to test thousands per week.”
How it works
With this process, the researchers engineered five distinct enzymes. The final pathway design comprises six total reaction steps, in which each enzyme performs one step. Together, the series of reactions successfully transformed formate into acetyl-CoA.
Much like the enzyme testing, the entire system is run outside of living cells. That means the team could precisely control enzyme concentrations, cofactors and conditions — something that’s nearly impossible to accomplish inside a living organism.
After establishing the system, Karim, Jewett and their teams used ReForm to convert acetyl-CoA into malate. The team also demonstrated ReForm can accept other carbon-based inputs, including formaldehyde and methanol.
“From here, we can imagine this work going in a couple different directions,” Karim said. “We would like to further optimize this pathway and explore other designs to make one-carbon conversions more efficient. We also can imagine using the tools that we developed to engineer all kinds of other new enzymes and pathways. It gives us hope for a future where we can combine multiple technologies, both biological and abiological, in unique ways to find new solutions.”
The study, “A synthetic cell-free pathway for biocatalytic upgrading of formate from electrochemically reduced carbon dioxide,” was supported by the U.S. Department of Energy (award number DE-SC0023278) and the National Science Foundation.
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