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Common Lab Bacteria Evolved to Use Just CO2 for Growth

Source: Weizmann Institute of Science

Source: Weizmann Institute of Science

Scientists at the Weizmann Institute of Science in Israel have generated strains of the bacterium Escherichia coli that consume just CO2 as their nutrient source, instead of organic compounds. They say that as well as demonstrating how adaptable the bacterial metabolism can be, the achievement could provide a foundation for harnessing synthetic biology to develop carbon-neutral bioproduction processes for food and fuels.

diagram researchers converted a common laboratory sugar eating E. coli bacterium
This diagram shows how researchers converted a common laboratory sugar eating (heterotrophic) E. coli bacterium (left) to produce all of its biomass from CO2 (autotrophic) by metabolic engineering combined with laboratory evolution. The new bacterium (center) uses the compound formate as a form of chemical energy to drive CO2 fixation by a synthetic metabolic pathway. The bacterium may provide the infrastructure for future industrial renewable production of food and green fuels (right). [Gleizer et al.]
“Our main aim was to create a convenient scientific platform that could enhance CO2 fixation, which can help address challenges related to sustainable production of food and fuels and global warming caused by CO2 emissions,” said research lead Ron Milo, PhD, a systems biologist at the Weizmann Institute of Science. “This feat is a powerful proof of concept that opens up a new exciting prospect of using engineered bacteria to transform products we regard as waste into fuel, food, or other compounds of interest … Converting the carbon source of E. coli, the workhorse of biotechnology, from organic carbon into CO2 is a major step towards establishing such a platform … It can also serve as a platform to better understand and improve the molecular machines that are the basis of food production for humanity and thus help in the future to increase yields in agriculture.”

The Weizmann team reports on its achievement in Cell, in a paper titled, “Conversion of Escherichia coli to Generate All Biomass Carbon from CO2.”

The living world is divided primarily into autotrophs—such as green plants, or algae—that convert inorganic CO2 into biomass, and heterotrophs—including humans and other animals—that consume organic compounds. In fact, autotrophic organisms “dominate the biomass on Earth,” the authors explained, “supplying all of our food and most of our fuel.”
A better understanding of the principles of autotrophic growth and potentially how to enhance autotrophic pathways could be critical for efforts to enhance sustainability. A “grand challenge in synthetic biology,” the investigators stated, has been the ability to generate synthetic autotrophy within a model heterotrophic organism. “In spite of widespread interest in renewable energy storage and more sustainable food production, the engineering of industrially relevant heterotrophic model organisms to use CO2 as their sole carbon source has so far remained an outstanding challenge.”

Achieving the “formidable task” of converting a heterotroph into a full autotroph requires that the host must be able to operate CO2 fixation pathways for which the carbon input is comprised solely of CO2, and that they are capable of producing from CO2 all of the precursor molecules needed to generate biomass. This requires expression of all the necessary enzymatic machinery for both production, regulation, and coordination of the autotrophic pathways. To date, the researchers noted, attempts to establish autocatalytic CO2 fixation cycles in model heterotrophs have always required the addition of multi-carbon organic compounds to achieve stable growth.

“From a basic scientific perspective, we wanted to see if such a major transformation in the diet of bacteria—from dependence on sugar to the synthesis of all their biomass from CO2—is possible,” stated first author Shmuel Gleizer, a Weizmann Institute of Science postdoctoral fellow. “Beyond testing the feasibility of such a transformation in the lab, we wanted to know how extreme an adaptation is needed in terms of the changes to the bacterial DNA blueprint.”

For the studies reported in Cell, the researchers effectively carried out some metabolic rewiring and used lab evolution to convert E. coli into autotrophs. “Our engineered E. coli strain uses the Calvin-Benson-Bassham cycle (CBB, also referred to as Calvin cycle for short) for carbon fixation,” they explained. The autotrophic strain harvests energy and reducing power from the one-carbon molecule formate (HCOO–), which can be produced electrochemically from renewable resources, which readily gives up the necessary electrons. Importantly, the formate doesn’t itself serve as a carbon source for E. coli growth, and doesn’t support heterotrophic pathways.

The researchers also engineered the bacteria to produce non-native enzymes for carbon fixation and reduction and for harvesting energy from the formate. But these changes were not on their own enough to support autotrophy, because E. coli‘s metabolism is adapted to heterotrophic growth. To overcome this challenge the researchers turned to adaptive laboratory evolution as a metabolic optimization tool. They inactivated central enzymes involved in heterotrophic growth, rendering the bacteria more dependent on autotrophic pathways for growth. Effectively, the bacteria were gradually weaned off the sugar they were used to consuming. At each stage, the cultured bacteria, grown in cheomstats, were given just enough sugar (xylose) to keep them from complete starvation, as well as plenty of CO2 and formate.

The investigators demonstrated that in this environment, there is a large selective advantage for autotrophs that produce biomass from CO2 as the sole carbon source, compared with heterotrophs that depend on xylose as a carbon source for growth. As some strains adapted to develop a taste for CO2 (giving them an evolutionary edge over those that still required sugar), their descendants were given less and less sugar until after about a year of adapting to the new diet some of them eventually made the complete switch, living and multiplying in an environment that served up pure CO2.

“… instead of attempting to rationally design components that comply with all the possible constraints, we created a rewired metabolic configuration and applied selective conditions under which the desired metabolic function is linked to fitness,” the team explained. “In order for the general approach of lab evolution to succeed, we had to find a way to couple the desired change in cell behavior to a fitness advantage,” Milo added. “That was tough and required a lot of thinking and smart design.”

To verify whether the bacteria were not somehow utilizing other nutrients for growth, some of the evolved E. coli were fed CO2 containing the heavy isotope C13. Then, the bacterial body parts were weighed, and the weight they had gained checked against the mass that would be added from eating the heavier version of carbon. The analysis showed the carbon atoms in the body of the bacteria were all extracted directly from CO2 alone.

By sequencing the genome and plasmids of the evolved autotrophic cells, the researchers discovered that as few as 11 mutations were acquired through the evolutionary process in the chemostat. One set of mutations affected genes encoding enzymes linked to the carbon fixation (Calvin) cycle. The second category consisted of mutations found in genes commonly observed to be mutated in previous adaptive laboratory evolution experiments, suggesting that they are not necessarily specific to autotrophic pathways. The third category consisted of mutations in genes with no known characterized role, which may be “hitchhiker mutations,” they stated. “Across the different isolates, there are anywhere between 2 and 27 extra mutated genes, some of which could be refinement mutations of the autotrophic phenotype but are not strictly essential for it. Future research into the genetic underpinning of the autotrophic phenotype will help determine which of the observed mutations is essential and sufficient for synthetic autotrophy.”

“The study describes, for the first time, a successful transformation of a bacterium’s mode of growth,” Gleizer said. “Teaching a gut bacterium to do tricks that plants are renowned for was a real long shot. When we started the directed evolutionary process, we had no clue as to our chances of success, and there were no precedents in the literature to guide or suggest the feasibility of such an extreme transformation. In addition, seeing, in the end, the relatively small number of genetic changes required to make this transition was surprising.” The authors acknowledged that one major study limitation of the technology as reported is that the consumption of formate by bacteria releases more CO2 than is consumed through carbon fixation. In addition, more research is needed before it’s possible to consider the scalability of the approach for industrial use.

Cell culture is now routinely used to produce commodity chemicals. It is feasible that such cells—yeast or bacteria—could be induced to live on a diet of CO2 and renewable electricity, negating the need to supply them with the large amounts of corn syrup they live on today. Bacteria could be further adapted so that rather than taking their energy from chemical substances such as format, they may be able to harness electrons directly from a solar collector, say, and then store that energy for later use as fuel in the form of carbon fixed in their cells. Such fuel would be carbon-neutral if the source of its carbon was atmospheric CO2.

The researchers will aim to continue developing the technology so that they can supply energy through renewable electricity to address the problem of CO2 release, determine whether ambient atmospheric conditions could support autotrophy, and try to narrow down the most relevant mutations for autotrophic growth. They hope that their work could springboard R&D that will lead to sustainable biomanufacturing in areas including food production. “The innovative potential of synthetic biology has led to an explosion of interest in leveraging recent advances toward sustainability challenges,” they stated. “One of the most important challenges is the assimilation of atmospheric CO2 for the production of food, fuels, and biochemicals.”

“Our lab was the first to pursue the idea of changing the diet of a normal heterotroph (one that eats organic substances) to convert it to autotrophism (‘living on air’),” says Milo. “It sounded impossible at first, but it has taught us numerous lessons along the way, and in the end, we showed it indeed can be done. Our findings are a significant milestone toward our goal of efficient, green scientific applications.”