Metabolic adaptation to TCA cycle deficiency in a strictly aerobic bacterium

Abstract The tricarboxylic acid (TCA) cycle is central to cellular metabolism, yet its disruption offers an attractive opportunity to redirect carbon toward biotechnological products. A key challenge is that severe TCA cycle interruption often results in auxotrophic phenotypes, limiting their practical application. Here, we used adaptive laboratory evolution to restore growth of the strictly aerobic bacterium Acinetobacter baylyi ADP1 following major disruption of the TCA cycle during growth on glucose minimal medium. Evolved strains recovered rapid growth through extensive reorganization of central metabolism, including reduced flux through the oxidative branch of the TCA cycle, enhanced anaplerotic cycling, increased acetate overflow, and remodeling of oxidative phosphorylation. Adaptation involved coordinated changes in quinone-linked electron transport and intracellular redox balancing, enabling continued growth despite reduced respiratory efficiency. These results demonstrate that adaptive evolution can overcome fundamental physiological constraints imposed by TCA cycle disruption and establish a framework for engineering carbon-partitioning phenotypes in aerobic production hosts.