After five years, the Li Yin team of the Institute of Microbiology has created a cycle that minimizes artificial carbon sequestration

The rapid development of industry has led to the continuous increase of carbon dioxide and other greenhouse gas emissions, prompting governments of all countries to accelerate the development of carbon dioxide capture and utilization technologies, and strive to achieve "carbon neutrality" as soon as possible
.

Among them, the design and creation of enzymes, biochemical pathways, engineered organisms or microbiomes with high-efficiency biological carbon fixation capacity has become an international research hotspot in the field of synthetic biological carbon fixation
.

In nature, plants and microorganisms can use six natural carbon fixation pathways to convert carbon dioxide into organic matter.
(Rubisco) is the Calvin cycle of the core carbon fixation enzyme
.

In order to break through the low efficiency of natural carbon fixation pathways, in 2016 and 2021, Science magazine successively reported the artificially designed non-natural carbon fixation pathway CETCH cycle (completed by the Max Planck Institute of Terrestrial Microbiology in Germany) and ASAP pathway (by the Chinese Academy of Sciences Tianjin).
Completed by the Industrial Biotechnology Research Institute), carbon dioxide can be converted into glyoxylic acid and starch in a cell-free system
.

The common feature of these natural and artificial carbon fixation pathways is that they are relatively long, generally involving more than ten biochemical reactions
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In general, the more reaction steps in the biochemical pathway, the lower the overall efficiency
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However, must the biological carbon fixation pathway consist of many reaction steps? Is it possible to design an artificial carbon fixation pathway with fewer reactions than the natural carbon fixation pathway? If it can be designed, under what conditions does such an artificial carbon fixation method need to operate, and how high a carbon fixation efficiency can be achieved? On December 28, 2021, the team of Researcher Li Yin from the Institute of Microbiology of the Chinese Academy of Sciences (Ph.
D student Xiao Lu is the first author) published a research paper titled A Minimized Synthetic Carbon Fixation Cycle in the ACS Catalysis journal
.

The research team lasted 5 years to design a new and minimized artificial carbon sequestration cycle
.

This cycle contains only four reactions, which are respectively catalyzed by pyruvate carboxylase (PYC), oxaloacetate acetyl hydrolase (OAH), acetate-CoA ligase (ACS) and pyruvate synthase (PFOR).
Named as POAP cycle (see figure below)
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Among the four-step reactions, the two reactions catalyzed by pyruvate synthase and pyruvate carboxylase are both carbon fixation reactions
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Each time the POAP cycle runs, two molecules of carbon dioxide can be converted to one molecule of oxalic acid, which consumes two molecules of ATP and one molecule of reducing power
.

The POAP cycle composed of four-step biochemical reactions.
Among the four-step reactions of the POAP cycle, the most critical and difficult to achieve is the reductive carboxylation catalyzed by pyruvate synthase (PFOR)
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Under normal circumstances, the reaction that we can generally observe in organisms is that PFOR catalyzes the oxidative decarboxylation of pyruvate and releases carbon dioxide
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To achieve the POAP cycle, it is necessary to reverse the oxidative decarboxylation catalyzed by PFOR, that is, to catalyze the reductive carboxylation of acetyl-CoA to produce pyruvate
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This reductive carboxylation reaction is thermodynamically unfavorable and requires a large reducing force to promote the reaction
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Therefore, researchers consider using low-potential electron donors to provide sufficient reducing power to drive the reaction under anaerobic conditions
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By synthesizing and testing a series of low-potential electron donor Ferredoxin (Ferredoxin, Fd), the researchers found that Fd2 from Hydrogenobacter thermophiles has a strong driving effect on the reductive carboxylation of PFOR
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Using Fd2 as an electron donor can drive the heterotetrameric pyruvate synthase derived from Clostridium thermocellum (Clostridium thermocellum) to realize the reductive carboxylation of acetyl-CoA
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This is also the first time that heterotetrameric pyruvate synthase has achieved reductive carboxylation in vitro
.

Through further synthetic tests, the researchers obtained the other three enzymes PYC, OAH and ACS needed to construct the POAP cycle
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Then adopt the strategy of constructing the POAP half-cycle separately and then integrating the two POAP half-cycles together to construct a functional POAP cycle
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Tested with 13C-labeled sodium bicarbonate as a substrate, and successfully detected 13C-labeled oxalic acid, the product of the POAP cycle, and determined that the carbon dioxide fixation rate of the POAP cycle was 8.
0±1.
8 nmol CO2/min/mg carbon fixation enzyme.
The conversion number is 5 mol/mol POAP cycle enzyme, and the carbon fixation efficiency is higher
.

In theory, three steps are required to form a minimum carbon fixation cycle
.

The POAP cycle contains only four reactions, which is close to the theoretical minimum, and it is also the smallest artificial carbon sequestration cycle that has been verified by experiments
.

Due to the short path, when the PFOR activity is much lower than the CETCH cycle carbon fixation enzyme activity, the carbon dioxide fixation rate of the POAP cycle still exceeds that of the CETCH cycle containing twelve-step reactions.

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The POAP cycle can achieve carbon dioxide fixation under anaerobic and higher temperature (50°C).
It provides a new model for understanding and studying how the early organisms carried out carbon dioxide fixation on the earth, and also provides a new model for the artificial biotransformation of carbon dioxide.
Optional way
.

Link to the paper: https://pubs.
acs.
org/doi/full/10.
1021/acscatal.
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