The assimilation of one-carbon (C1) compounds, such as methanol, by serine cycle methylotrophs requires the continuous regeneration of glyoxylate. the determination of the overall topology of its metabolic network. The operation of the ethylmalonyl-CoA pathway in AM1 has major implications for the physiology of these methylotrophs and their role in nature, and it also provides a common ground for C1 and C2 compound assimilation in isocitrate lyase-negative bacteria. AM1, one of the most studied methylotrophs, has been a longstanding goal, and although great progress has been made (2C5), it is still not fully achieved. A key point has been to understand how the bacterium incorporates C1 units into cell material. The serine cycle was elucidated in this organism during the early 1960s by Quayle and coworkers (6C9). The assimilation of C1 units by this pathway requires continuous regeneration of glyoxylate from acetyl-CoA and can be achieved, in principle, via the well-known glyoxylate cycle (10). However, Dunstan and coworkers (11C14) showed in 1972 and 1973 that AM1 lacks the key enzyme of the glyoxylate cycle, isocitrate lyase, but has an alternative route involving oxidation of acetate to glyoxylate that functions during growth on both C1 and C2 compounds. Also, other organisms, including the photosynthetic are known to require an alternative to the glyoxylate cycle when growing on C2 substrates or on substrates that are converted into acetyl-CoA to enter central metabolism (15C18). Recent studies, including mutant analyses, gene predictions, enzyme assays, and metabolite studies in AM1, have led to the observation that a complex sequence of CoA thioester derivatives is involved in glyoxylate regeneration, resulting in the hypothesis of the so-called glyoxylate regeneration cycle (GRC) (19, 20) [Fig. 1 and supporting information (SI) 5142-23-4 manufacture Table S1]. According to this pathway, a C5 compound, methylsuccinyl-CoA, is formed from the condensation of 2 acetyl-CoA molecules plus 1 CO2 and is decarboxylated twice in a process similar to valine degradation. The specific intermediates of the GRC are isobutyryl-CoA, metacrylyl-CoA, and hydroxyisobutyryl-CoA, and the result is the formation of propionyl-CoA. Subsequently, propionyl-CoA is transformed to malate, from which 1 glyoxylate and 1 acetyl-CoA are generated (20). More recently, 5142-23-4 manufacture a second hypothesis, 5142-23-4 manufacture the ethylmalonyl-CoA pathway (EMCP), was proposed from studies of C2 assimilation pathways in (21C23). This pathway (Fig. 1 and Table S1) includes the formation of methylsuccinyl-CoA, which is further converted to methylmalyl-CoA, from which both glyoxylate and propionyl-CoA are released by cleavage (22). The propionyl-CoA can then be converted to C4 compounds and assimilated as cell material (23). Fig. 1. Pathways proposed for glyoxylate regeneration in isocitrate lyase-negative bacteria. The reactions that are specific to the GRC (20) or to the EMCP (23) are indicated. For designations of genes and enzymes, see Table S1. Metabolite numbers are according … The 2 2 pathways mentioned above are still hypothetical, and none has been firmly demonstrated to operate in vivo. They differ strikingly in terms of carbon balance and, therefore, INMT antibody overall carbon yield for methylotrophic growth. The GRC includes a net decarboxylation step, whereas the ethylmalonyl-CoA pathway includes net carboxylation steps. This makes the second pathway more efficient in terms of carbon assimilation and has important implications with regard to the physiology of these methylotrophs and their actual biotechnological potential. In this work, we combined state-of-the-art metabolomics.