Formate can be directly produced from CO₂ and renewable electricity, making it a promising microbial feedstock for sustainable bioproduction. Cupriavidus necator is one of the few biotechnologically-relevant hosts that can grow on formate, but it uses the Calvin cycle, the high ATP cost of which limits biomass and product yields. Here, we redesign C. necator metabolism for formate assimilation via the synthetic, highly ATP-efficient reductive glycine pathway. First, we demonstrate that the upper pathway segment supports glycine biosynthesis from formate. Next, we explore the endogenous route for glycine assimilation and discover a wasteful oxidation-dependent pathway. By integrating glycine biosynthesis and assimilation we are able to replace C. necator's Calvin cycle with the synthetic pathway and achieve formatotrophic growth. We then engineer more efficient glycine metabolism and use short-term evolution to optimize pathway activity. The final growth yield we achieve (2.6 gCDW/mole-formate) nearly matches that of the WT strain using the Calvin Cycle (2.9 gCDW/mole-formate). We expect that further rational and evolutionary optimization will result in a superior formatotrophic C. necator strain, paving the way towards realizing the formate bio-economy.

甲酸盐可以直接从 CO ₂和可再生电力中生产，使其成为可持续生物生产的有前途的微生物原料。Cupriavidus necator是少数可以在甲酸盐上生长的生物技术相关宿主之一，但它使用卡尔文循环，其高 ATP 成本限制了生物量和产品产量。在这里，我们通过合成的、高效的 ATP 还原甘氨酸途径重新设计了C. necator代谢以进行甲酸同化。首先，我们证明了上游途径片段支持从甲酸进行甘氨酸生物合成。接下来，我们探索甘氨酸同化的内源性途径，并发现了一种浪费的氧化依赖性途径。通过整合甘氨酸生物合成和同化，我们能够取代C. necator的卡尔文循环与合成途径并实现形态营养生长。然后，我们设计了更有效的甘氨酸代谢，并使用短期进化来优化通路活性。我们实现的最终生长产量（2.6 gCDW/摩尔甲酸盐）几乎与使用卡尔文循环的 WT 菌株（2.9 gCDW/摩尔甲酸盐）相匹配。我们预计进一步的理性和进化优化将产生优良的形态营养型C. necator菌株，为实现甲酸生物经济铺平道路。