Researchers Engineer Biosynthetic Pathway for More Effective Carbon Fixation

It is a known fact that different varieties of organisms on earth extract enzymes to convert CO₂ into organic compounds such as sugars (e.g. plants convert CO₂ via photosynthesis). However, attempts at exploiting such potentials to convert CO₂ into products of high value, e.g. renewable chemicals and biofuel, have not been successful enough. Although the increasing concentration of atmospheric CO₂ is an issue, scientists view it as an opportunity.

A group of researchers at the Max Planck Institute (MPI) for Terrestrial Microbiology in Marburg, Germany, have leveraged the DNA synthesis expertise of the U.S. Department of Energy Joint Genome Institute (DOE JGI) to reverse engineer a biosynthetic pathway to enable more effective carbon fixation.

The innovative pathway is formed with a new CO₂-fixing enzyme that can convert CO₂ close to 20 times faster than the most prevalent natural enzyme that captures CO₂ in plants by making use of sunlight as energy. The research work was reported in the journal Science, in the 18 November, 2016 issue.

We had seen how efforts to directly assemble synthetic pathways for CO₂-fixation in a living organism did not succeed so far. So we took a radically different, reductionist approach by assembling synthetic principal components in a bottom-up fashion in a test tube.

Tobias Erb, MPI

The group started by working on multiple theoretical CO₂-fixation paths that could lead to continuous carbon cycling. However, they did not discontinue the study there. “We did not restrict our design efforts to known enzymes, but considered all reactions that seemed biochemically feasible,” Erb said.

In contrast to DNA sequencing in which the concept of life is perceived from an organism’s genome, DNA synthesis involves identifying a specific genetic element (e.g. an enzyme for assimilating atmospheric carbon) at first, and then writing and expressing the specific code into a new system.

As a conclusion, by way of sequencing and synthesis, the researchers sourced 17 disparate enzymes from 9 disparate organisms from the three kingdoms of life and organized them to accomplish a proof of principle CO₂-fixation pathway performance that surpasses the natural one. According to Erb, this is the “CETCH cycle” for crotonyl-CoA/ethylmalonyl-12 CoA/hydroxybutyryl-CoA since it efficiently “cetches” atmospheric CO₂.

The researchers utilized the idea of metabolic “retrosynthesis,” e.g. breaking down the reaction step by step beginning from the smaller precursors, and cleverly manipulated the thermodynamic conditions to invent a strategy that produces highly positive outcomes that competed well with naturally existing metabolic pathways.

Then, they thoroughly searched the public databases for enzymes that were closely related to their model and chose many to test them.

We first reconstituted its central CO₂-fixation reaction sequence stepwise, providing the ingredients to catalyze all the desired reactions. Then, by following the flux of CO₂ we discovered which particular key reaction was rate-limiting.

This resulted in methylsuccinyl-CoA dehydrogenase (Mcd), part of a group of enzymes responsible for respiration, i.e. the metabolic reaction that takes place in the cells of living things to convert carbon into energy.

To overcome this limitation, we engineered the Mcd to use oxygen as an electron acceptor, to amp up the function, but this was not quite enough. We had to replace the original pathway design with alternative reaction sequences, used further enzyme engineering to minimize side reactions of promiscuous enzymes, and introduced proofreading enzymes to correct for the formation of dead-end metabolites.

Tobias Erb, MPI

To assist the MPI group’s research, hundreds of Enoyl-CoA Carboxylase/Reductase (ECR) enzyme variants were synthesized by the DOE JGI via its Community Science Program, allowing the MPI group to find the ECR with the greatest CO₂-fixation activity to successfully form a highly efficacious artificial CO₂-fixation pathway in a test tube.

“ECRs are supercharged enzymes that are capable of fixing CO₂ at the rate of nearly 20 times faster than the most widely prevalent CO₂-fixing enzyme in nature, RuBisCo, which carries out the heavy lifting involved in photosynthesis,” Erb said.

The chemical process uses sunlight to convert CO₂ into sugars that can be utilized by cells as energy, together with different natural processes on earth, and includes the conversion of nearly 350 billion tons of CO₂ in a year.

Seventy years ago, this phenomenon interested early Berkeley Lab researcher Melvin Calvin. Calvin and his collaborators Andrew Benson and James Bassham described the cycle in plants, algae, and microorganisms, and it bears their names. Calvin was awarded the Nobel Prize for the same in the year 1961.

The current generation of researchers are working toward assimilating excess CO₂ from the atmosphere and converting it into energy and natural products to boost the economy.

Now Berkeley Lab through the DOE Joint Genome Institute, has been a major contributor to our understanding of the vast genetic diversity of microorganisms and their roles in the environment, particularly in carbon cycling. By sequencing underexplored phyla from ecologically important niches, we have homed in on the genes and pathways that we now are able to synthesize in the lab to unravel novel strategies that nature uses for carbon metabolism. Identifying these genes encoding CO₂-fixing enzymes and their biological function, is one of the important missing pieces in the climate puzzle.

Yasuo Yoshikuni, Head of the DNA Synthesis Science, DOE JGI

Encouraged by the triumphant simulation of a synthetic enzymatic network in a test tube for transforming CO₂ into organic products, which is far better than natural chemical processes, Erb said this will pave the way for other future endeavors.

“These could include the introduction of synthetic CO₂-fixation cycles into organisms to bolster natural photosynthesis, or say, in combination with photovoltaics, lead the way to artificial photosynthesis, this might at the end jumpstart the design of self-sustaining, completely synthetic carbon metabolism in bacterial and algal systems.”

Yoshikuni looks forward to the prospects of converging DNA sequencing and biological functions further, thereby influencing DNA synthesis. “Through DOE JGI’s high-throughput sequencing capabilities coupled with the rapidly decreasing price of DNA synthesis, we continue to enable our user community in bringing to light the physiological potential of microorganisms and microbial communities. In the longer term, we hope to expect to see these test-tube results yield a new generation of real bioproducts delivered to address critical energy and environmental challenges.”

The wider implication of this research is to effectively elucidate the enhanced role of “engineering thinking” in biotechnology, since the increased characterization of the biological “parts list” resulting at the end of high throughput genome sequencing provides higher chances of reconstructing by designing abilities in living organisms that can fulfill the requirements of the DOE mission in the fields of environment and bioenergy.

The Max-Planck Society, the European Research Council, ETH Zurich, and the Swiss National Science Foundation supported this research. DOE JGI is a DOE Office of Science User Facility.