The reductive glycine pathway was described as the most energetically favorable synthetic route of aerobic formate assimilation. Here we report the successful implementation of formatotrophy in Escherichia coli by means of a stepwise adaptive evolution strategy. Medium swap and turbidostat regimes of continuous culture were applied to force the channeling of carbon flux through the synthetic pathway to pyruvate establishing growth on formate and CO₂ as sole carbon sources. Labeling with ¹³C-formate proved the assimilation of the C1 substrate via the pathway metabolites. Genetic analysis of intermediate isolates revealed a mutational path followed throughout the adaptation process. Mutations were detected affecting the copy number (gene ftfL) or the coding sequence (genes folD and lpd) of genes which specify enzymes implicated in the three steps forming glycine from formate and CO₂, the central metabolite of the synthetic pathway. The mutation R191S present in methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) abolishes the inhibition of cyclohydrolase activity by the substrate formyl-tetrahydrofolate. The mutation R273H in lipoamide dehydrogenase (Lpd) alters substrate affinities as well as kinetics at physiological substrate concentrations likely favoring a reactional shift towards lipoamide reduction. In addition, genetic reconstructions proved the necessity of all three mutations for formate assimilation by the adapted cells. The largely unpredictable nature of these changes demonstrates the usefulness of the evolutionary approach enabling the selection of adaptive mutations crucial for pathway engineering of biotechnological model organisms.

还原甘氨酸途径被描述为有氧甲酸盐同化的最有利的合成途径。在这里，我们报告了通过逐步适应性进化策略在大肠杆菌中成功实施格式营养。连续培养的培养基交换和浊度调节方案被用于强制碳通量通过合成途径引导到丙酮酸，从而在甲酸盐和 CO ₂作为唯一碳源上建立生长。^用13 C-甲酸盐标记证明了 C1 底物通过途径代谢物同化。中间分离物的遗传分析揭示了在整个适应过程中遵循的突变路径。检测到影响拷贝数的突变（基因ftfL) 或基因的编码序列（基因folD和lpd），这些基因指定了与从甲酸和 CO _{2形成甘氨酸的三个步骤有关的酶}，合成途径的中心代谢物。存在于亚甲基-四氢叶酸脱氢酶/环化水解酶 (FolD) 中的突变 R191S 消除了底物甲酰基-四氢叶酸对环化水解酶活性的抑制作用。硫辛酰胺脱氢酶 (Lpd) 中的突变 R273H 改变了底物亲和力以及生理底物浓度下的动力学，可能有利于向硫辛酰胺还原的反应转变。此外，基因重建证明了适应细胞对甲酸同化的所有三种突变的必要性。这些变化在很大程度上不可预测的性质证明了进化方法的有用性，它能够选择对生物技术模型生物的途径工程至关重要的适应性突变。