Dynamic adaptive responses, degradation pathway, and iron-mediated enhancement in 17β-estradiol biodegradation by cold-exposed Rhodococcus sp. RCBS9

The molecular mechanism by which microorganisms degrade the endocrine-disrupting contaminant 17β-estradiol (E2) at low temperatures remains largely unclear, with only limited studies available. Here, we investigated the adaptive strategy of the cryotolerant bacterium Rhodococcus sp . RCBS9 for sustaining E2 degradation at 10 °C, a temperature at which efficient estrogen degradation has rarely been reported. Using time-resolved multi-omics and phenotypic assays, we characterized a dynamic, staged adaptation process involving changes in oxidative stress defense, membrane composition, transcription factors, signaling factors, and stress proteins. We uncovered three major novelties. First, RCBS9 employs a distinct degradation pathway that first cleaves the saturated sterol rings, in contrast to the canonical 4,5-seco pathway that initiates with aromatic ring A cleavage. Second, we identified and functionally validated a cold-tolerant short-chain dehydrogenase/reductase (SDR) encoded by gene RQCS_RS03565 that converts E2 to estrone (E1), as confirmed by heterologous expression and site-directed mutagenesis. Third, subsequent sterol ring cleavage proceeds via a suite of oxidoreductases and hydrolases. The resulting aromatic intermediates are then mineralized through the β-ketoadipate pathway, as suggested by the coordinated upregulation of intradiol dioxygenase and carboxymuconolactone decarboxylase. Additionally, trace iron (50 μM) promoted E2 degradation and reduced E1 accumulation, partly attributable to enhanced metabolic activity and upregulation of iron-requiring enzyme genes. Overall, this work provides fundamental insights into a novel estrogen biodegradation logic under cold stress and proposes a practical iron-mediated strategy for low-temperature bioremediation.