Purple phototrophic bacteria release crotonate as metabolic overflow pathway to complement other redox-balancing routes

Purple phototrophic bacteria (PPB) are renowned for versatile metabolic strategies to maintain redox balance, including CO₂ fixation, H₂ production, and polymer storage. Here we report a previously unknow redox-balancing mechanism in PPB: the extracellular release of crotonate as a metabolic overflow. In batch photoheterotrophic cultures of enriched PPB, crotonate accumulated transiently under conditions of carbon excess. Crotonate excretion coincided with the depletion of other electron sinks (polyhydroxybutyrate and H₂) and was reversed when an alternate electron acceptor (DMSO) was provided, indicating a regulated overflow rather than irreversible fermentation. Using metaproteomics, we found that dominant Rhodopseudomonas species redirect metabolism toward crotonyl-CoA production when conventional acetyl-CoA assimilation routes are limited. Key enzymes of the glyoxylate cycle were suppressed, while enzymes converting acetyl-CoA to crotonyl-CoA were up-regulated, leading to crotonyl-CoA accumulation. Notably, a CoA-transferase enzyme was identified as a candidate responsible for converting crotonyl-CoA to free crotonate, enabling excretion. These results reveal crotonate as an “escape valve” metabolite that PPB naturally deploy to dissipate excess reductant. The discovery of crotonate overflow expands our understanding of microbial redox homeostasis and highlights a novel facet of PPB metabolism with potential implications for optimizing biotechnological processes such as bioplastic production