2A). First, we focused on the initial oxidation of glycolate to glyoxylate, which is the first step in all glycolate-metabolizing routes. RNA was isolated during midlog phase from C. necator grown in shake-flask cultures on minimal medium (JMM) with a headspace consisting of ambient air and 4% hydrogen or 10% CO2 and 4% hydrogen. Serine is then deaminated to hydroxypyruvate, further reduced to glycerate, and finally phosphorylated to generate the Calvin cycle intermediate 3PG. Rather, the glyoxylate produced in phosphoglycolate salvage can be condensed with acetyl-CoA generated from the carbon fixation process, and the resulting malate is assimilated into biomass (i.e., via the tricarboxylic acid [TCA] cycle, cataplerosis, and gluconeogenesis). The reactors were stirred at 200 rpm, and temperature was controlled at 30 °C. For example, 3 RuBP C5 indicates that there are 3 RuBP molecules present, each containing 5 carbon atoms. Growth on oxalate can proceed via two routes (SI Appendix, Fig. However, the presence of malate synthase has been confirmed in some cyanobacteria, leading to the suggestion that they may use the malate cycle (40⇓⇓–43). Those chemolithoautotrophs that employ the Calvin cycle under aerobic conditions must also cope with the oxygenase side activity of Rubisco by recycling or removing 2PG. We leave it for future studies to explore the occurrence of the malate cycle and other phosphoglycolate salvage routes in cyanobacteria and chemolithoautotrophs. It is a cyclic biochemical pathway which proceeds with the use of carbon dioxide and the production of oxygen. Precultures for these experiment were performed in JMM with 20 mM fructose in glass tubes, and next, they were washed three times and inoculated at an OD600 (optical density at 600 nm) of 0.01 or 0.005; 150 μL of culture medium was topped with 50 μL of transparent mineral oil (Sigma-Aldrich) to prevent evaporation (O2 and CO2 can diffuse well through this oil). Stage 2: Reduction Using the energy from ATP, the three-carbon compound, 3-PGA molecules, produced in the carbon fixation stage, are converted into a three-carbon sugar glyceraldehyde-3-phosphate (G3P). This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism. However, of the remaining ∼1,900 noncyanobacterial genomes found to encode Rubisco, ∼60% also encode a malate synthase. Where does the Calvin cycle occur mastering biology. Biological duplicates of each knockout strain were constructed. We show that the C2 cycle and decarboxylation via oxalate do not support phosphoglycolate salvage in C. necator. were funded by the Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy–EXC 2008–390540038–UniSysCat. Author contributions: N.J.C. The reactions of the Calvin cycle add carbon (from carbon dioxide in the atmosphere) to a simple five-carbon molecule called RuBP. Edited by Donald R. Ort, University of Illinois at Urbana–Champaign, Urbana, IL, and approved July 24, 2020 (received for review June 14, 2020). To determine the relative importance of each candidate pathway in phosphoglycolate salvage, we tested wild-type C. necator and several of the gene deletion strains described above for their ability to grow autotrophically at ambient CO2 (Fig. In light of recent results, they’re not so sure. This project was funded by the Max Planck Society. Copyright © 2020 the Author(s). The Calvin cycle takes place inside the chloroplasts of leaves. Candidate pathways supporting phosphoglycolate salvage in C. necator; 2PG is first dephosphorylated to glycolate and then oxidized, by the glycolate dehydrogenase complex, to give glyoxylate. S1): 1) assimilation to central metabolism via the glycerate pathway, which depends on the activity of a CoA-acylating glyoxylate dehydrogenase (in the reverse direction of that required for growth on glycolate), and 2) complete oxidation of oxalate, which can be followed by carbon fixation via the Calvin cycle. Finally, the Synechocystis-like oxalate decarboxylation pathway seems not to be present in C. necator, as no oxalate decarboxylase gene could be found. Explore more about C3 cycle or Calvin cycle and other stages of Calvin cycle @ BYJU'S Growth experiments on ambient CO2 were performed in biological duplicates and showed identical growth curves (±5%); hence, representative curves of a single experiment are shown. 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