Acetate utilization strategy in chain-elongating bacteria determines butyrate versus medium-chain carboxylate...

Chain-elongating bacteria (CEB) are a unique guild of anaerobes that upcycle organic waste into valuable short- and medium-chain carboxylic acids (MCCAs), enabling a circular bioeconomy. However, the metabolic rules that determine product chain length have remained elusive. Here we combine ¹³C isotope tracing, proteomics, enzyme assays and metabolic modelling to show that distinct acetate utilization strategies underlie the divergence between MCCA-producing CEB and those solely producing less valuable, short-chain butyrate. MCCA-producing strains recycle acetate to maximize lactate use under acetate limitation, but at the cost of slower growth. In contrast, butyrate-producing strains grow faster by favouring acetate assimilation, at the cost of restricted lactate utilization when acetate is scarce. These physiological trade-offs are encoded in the substrate specificity of coenzyme A transferase, the terminal enzyme in reverse β-oxidation. Our findings uncover a fundamental constraint shaping chain-length selectivity in CEB and offer strategies that could optimize MCCA production from organic waste streams.