biostudies-literatureDiehl CLOEWE program state of HesseGordon and Betty Moore FoundationMax-Planck-Gesellschaft168-175https://www.ebi.ac.uk/biostudies/studies/S-EPMC9889269biostudies-literatureUnknown19(2)Anaplerosis is an essential feature of metabolism that allows the continuous operation of natural metabolic networks, such as the citric acid cycle, by constantly replenishing drained intermediates. However, this concept has not been applied to synthetic in vitro metabolic networks, thus far. Here we used anaplerotic strategies to directly access the core sequence of the CETCH cycle, a new-to-nature in vitro CO₂-fixation pathway that features several C₃-C₅ biosynthetic precursors. We drafted four different anaplerotic modules that use CO₂ to replenish the CETCH cycle's intermediates and validated our designs by producing 6-deoxyerythronolide B (6-DEB), the C₂₁-macrolide backbone of erythromycin. Our best design allowed the carbon-positNature chemical biologySynthetic anaplerotic modules for the direct synthesis of complex molecules from CO₂.PMC9889269GBMF10652Erb TJDiehl CPaczia NGerlinger PDfalseSynthetic anaplerotic modules for the direct synthesis of complex molecules from CO₂.Anaplerosis is an essential feature of metabolism that allows the continuous operation of natural metabolic networks, such as the citric acid cycle, by constantly replenishing drained intermediates. However, this concept has not been applied to synthetic in vitro metabolic networks, thus far. Here we used anaplerotic strategies to directly access the core sequence of the CETCH cycle, a new-to-nature in vitro CO₂-fixation pathway that features several C₃-C₅ biosynthetic precursors. We drafted four different anaplerotic modules that use CO₂ to replenish the CETCH cycle's intermediates and validated our designs by producing 6-deoxyerythronolide B (6-DEB), the C₂₁-macrolide backbone of erythromycin. Our best design allowed the carbon-posit2023-01-01T00:00:00Z2023 Feb2025-04-21T22:52:19.782Z2025-04-05T18:57:23.084ZS-EPMC98892693647099410.1038/s41589-022-01179-0