Bacterial estrogenesis without oxygen: Wood–Ljungdahl pathway likely contributed to the emergence of estrogens in the biosphere

Androgen and estrogen, key sex hormones, were long thought to be exclusively produced by vertebrates. The O₂-dependent aromatase that converts androgen to estrogen (estrogenesis) has never been identified in any prokaryotes. Here, we report the finding of anaerobic estrogenesis in a Peptococcaceae bacterium (<i>Phosphitispora</i> sp. strain TUW77) isolated from the gut of the great blue-spotted mudskipper (<i>Boleophthalmus pectinirostris</i>). This strain exhibits testosterone fermentation pathways, transforming testosterone into estrogens and androstanediol under anaerobic conditions. Physiological experiments revealed that strain TUW77 grows exclusively on testosterone, utilizing the androgenic C-19 methyl group as both the carbon source and electron donor. The genomic analysis identified three copies of a polycistronic gene cluster, <i>abeABC</i> (anaerobic bacterial estrogenesis), encoding components of a classic cobalamin-dependent methyltransferase system. These genes, highly expressed under testosterone-fed conditions, show up to 57% protein identity to the characterized EmtAB from denitrifying <i>Denitratisoma</i> spp., known for methylating estrogen into androgen (the reverse reaction). Tiered transcriptomic and proteomic analyses suggest that the removed C-19 methyl group is completely oxidized to CO₂ via the oxidative Wood-Ljungdahl pathway (WLP), while the reducing equivalents (NADH) fully reduce remaining testosterone to androstanediol. Consistently, the addition of anthraquinone-2,6-disulfonate, an extracellular electron acceptor, to testosterone-fed TUW77 cultures enabled complete testosterone conversion into estrogen without androstanediol accumulation (anaerobic testosterone oxidation). This finding of aromatase-independent estrogenesis in anaerobic bacteria suggests that the ancient WLP may have contributed to the emergence of estrogens in the early biosphere.