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The metabolism of one- and two-carbon compounds by the methylotrophic bacteriumMethylobacterium extorquens AM1 involves high carbon flux through the ethylmalonyl coenzyme A (ethylmalonyl-CoA) pathway (EMC pathway). During growth on ethylamine, the EMC pathway operates as a linear pathway carrying the full assimilatory flux to produce glyoxylate, malate, and succinate. Assimilatory carbon enters the ethylmalonyl-CoA pathway directly as acetyl-CoA, bypassing pathways for formaldehyde oxidation/assimilation and the regulatory mechanisms controlling them, making ethylamine growth a useful condition to study the regulation of the EMC pathway. Wild-typeM. extorquens cells were grown at steady state on a limiting concentration of succinate, and the growth substrate was then switched to ethylamine, a condition where the cell must make a sudden switch from utilizing the tricarboxylic acid (TCA) cycle to using the ethylmalonyl-CoA pathway for assimilation, which has been an effective strategy for identifying metabolic control points. A 9-h lag in growth was observed, during which butyryl-CoA, a degradation product of ethylmalonyl-CoA, accumulated, suggesting a metabolic imbalance. Ethylmalonyl-CoA mutase activity increased to a level sufficient for the observed growth rate at 9 h, which correlated with an upregulation of RNA transcripts forecm and a decrease in the levels of ethylmalonyl-CoA. When the wild-type strain overexpressingecm was tested with the same substrate switchover experiment, ethylmalonyl-CoA did not accumulate, growth resumed earlier, and, after a transient period of slow growth, the culture grew at a higher rate than that of the control. These findings demonstrate that ethylmalonyl-CoA mutase is a metabolic control point in the EMC pathway, expanding our understanding of its regulation.

Ethylmalonyl Coenzyme A Mutase Operates as a Metabolic Control Point in Methylobacterium extorquens AM1