Anaplerosis, the replenishment of metabolic intermediates, is crucial for natural metabolic networks. This study applies anaplerotic strategies to a synthetic CO₂-fixation pathway (CETCH cycle) to directly access its core intermediates. Four anaplerotic modules, utilizing CO₂, were designed and validated by synthesizing 6-deoxyerythronolide B (6-DEB), the erythromycin backbone. The best design achieved carbon-positive 6-DEB synthesis via 54 enzymatic reactions *in vitro*, with yields comparable to isolated 6-DEB polyketide synthase (DEBS). This demonstrates the design and tailoring of anaplerotic modules to enhance synthetic capabilities of complex *in vitro* reaction networks.

Christoph Diehl, Patrick D. Gerlinger, Nicole Paczia, Tobias J. Erb