Genetically modified organisms (GMOs) may outgrow Frankenfood production. Soon, they may take over Frankenfixation, the industrial conversion of atmospheric carbon dioxide (CO2) to biomass. That’s the word from the U.S. Department of Energy’s Joint Genome Institute (JGI), which has overseen an effort to stitch together an artificial metabolism from the bits and pieces of biosynthetic pathways that were once scattered across the three kingdoms of life.

As part of the JGI project, scientists from the Max-Planck-Institute (MPI) have reverse engineered a novel pathway that is based on a new CO2-fixing enzyme that is nearly 20 times faster than the most prevalent enzyme in nature responsible for capturing CO2 in plants by using sunlight as energy.

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

The team started with several theoretical CO2-fixation routes that could result in continuous carbon cycling. But they didn't stop there. “We did not restrict our design efforts to known enzymes, but considered all reactions that seemed biochemically feasible,” noted Erb.

Unlike DNA sequencing, where the language of life is read from the genome of an organism, DNA synthesis entails first the identification of a particular genetic element—such as an enzyme for fixing carbon from the atmosphere—and writing and expressing that code in a new system.

In the end, they sourced, through sequencing and synthesis, 17 different enzymes from nine different organisms and assembled these parts to create a new CO2-fixation pathway. This pathway is the centerpiece of a proof-of-principle study (“A Synthetic Pathway for the Fixation of Carbon Dioxide In Vitro”). It appeared November 18 in the journal Science.

“The crotonyl–coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle is a reaction network of 17 enzymes that converts CO2 into organic molecules at a rate of 5 nanomoles of CO2 per minute per milligram of protein,” the study’s authors wrote. “The CETCH cycle was drafted by metabolic retrosynthesis and optimized in several rounds by enzyme engineering and metabolic proofreading.”

By “metabolic retrosynthesis,” the authors refer to how they dismantled the cycle’s components step by step. The MPI team also juggled the thermodynamic conditions and came up with a strategy that yielded more promising results that competed favorably with naturally occurring metabolic pathways. Then they plumbed the depths of the public databases for enzymes that would support their model and selected several dozen to try out.

“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 turned out to be methylsuccinyl-CoA dehydrogenase (Mcd), part of a family of enzymes involved in respiration—the metabolic reaction in the cells of organisms to convert nutrients like carbon into units of 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,” said Erb. “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.”

In support of the MPI team's efforts, the JGI synthesized hundreds of enoyl-CoA carboxylase/reductase (ECR) enzyme variants through its Community Science Program. This enabled the MPI team to zero in on the ECR with the highest CO2-fixation activity to successfully build a more efficient 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.

This chemical process harnesses sunlight to turn CO2 into sugars that cells can use as energy along with other natural processes on the planet and accounts for the transformation of some 350 billion tons of CO2 annually.

“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,” explained Yasuo Yoshikuni, the head of the DNA Synthesis Science group at the JGI. “Identifying these genes encoding CO2-fixing enzymes and their biological function is one of the important missing pieces in the climate puzzle.”

Emboldened by the successful reconstitution of a synthetic enzymatic network in a test tube for the conversion of CO2 into organic products that is superior to chemical processes and competes with favorably with those in nature, the authors of the Science paper anticipate future steps: “The CETCH cycle adds a seventh, synthetic alternative to the six naturally evolved CO2 fixation pathways, thereby opening the way for in vitro and in vivo applications.”

“These could include the introduction of synthetic CO2-fixation cycles into organisms to bolster natural photosynthesis,” Erb elaborated. Synthetic pathways “in combination with photovoltaics might lead the way to artificial photosynthesis, jumpstarting the design of self-sustaining, completely synthetic carbon metabolism in bacterial and algal systems.”

The broader significance of this work is to dramatically illustrate the increased role of “engineering thinking” in biotechnology as the accelerated characterization of the biological “parts list” emerging from high-throughput genome sequencing furnishes greater opportunities to reconstruct by design the capacities in living organisms that address DOE mission needs in bioenergy and environment.

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