Development of autotrophy in Escherichia coli through adaptive laboratory evolution

ABSTRACT Enabling heterotrophic Escherichia coli to use CO 2 as its only carbon source remains a great challenge, and previous studies approached autotrophy conversion by metabolic engineering. Although its native carbon fixation routes were identified, the potential to reach autotrophy by itself has long been overlooked. In this study, autotrophy in E. coli was developed through adaptive laboratory evolution. After 1,000 days of consecutive inorganic subculturing, missense mutations were found in isocitrate dehydrogenase icd and isocitrate dehydrogenase kinase/phosphatase aceK genes, determining the metabolic switch between the citrate cycle and the glyoxylate shunt. By transcriptomic comparison of the adapted E. coli between inorganic and organic cultivations, two CO 2 fixing enzymes activated in autotrophic mode were found, including the upregulated pyruvate:ferredoxin oxidoreductase YdbK and phosphoenolpyruvate carboxykinase Pck. Connected by the upregulated phosphoenolpyruvate synthase PpsA, a carbon fixation module was constituted, which was the shared foundation of the aspartate-threonine cycle and the citrate-glyoxylate-methylcitrate cycle, and thus integrating into an autotrophic network. By comparing the 13 C enrichment patterns in inorganic cultivations between the adapted and initial E. coli , the favorable direction of the autotrophic network was confirmed. IMPORTANCE This is the first study to accomplish autotrophy in E. coli through long-term evolution alone. Besides missense mutations in icd and aceK genes, adapted E. coli also actively regulated its gene expression to respond to inorganic environment, such as directing the metabolic switch towards the glyoxylate shunt. For biomass formation, a carbon fixation module consisted of the upregulated YdbK, PpsA, and Pck produced pyruvate and oxaloacetate as precursors for two cycles. The aspartate-threonine cycle with a replenishment side loop further accumulated these precursors, and the citrate-glyoxylate-methylcitrate cycle was driven by four overexpressed enzymes to catalyze six reactions. These metabolic pathways were integrated into a novel autotrophic network, and by understanding the nature of E. coli , rational designs for its carbon fixation optimization become attainable by using compatible mechanisms.