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

Abstract

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 (Phosphitispora sp. strain TUW77) isolated from the gut of the great blue-spotted mudskipper (Boleophthalmus pectinirostris). 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, abeABC (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 Denitratisoma 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.

Keywords: C1 metabolism; Wood–Ljungdahl pathway; anaerobic microbiology; aromatics; methyltransferase.

Fig. 1.

Identification of Phosphitispora sp. strain TUW77 and characterization of the anaerobic testosterone metabolism. (A) Scanning electron micrograph showing strain TUW77 cells isolated from mudskipper gut. (B) Fermentative growth of strain TUW77 with unlabeled testosterone. 19-Nortestosterone served as a reference compound (not metabolized by strain TUW77). Data represent means ± SEM (n = 3). UPLC–APCI–HRMS analysis of the testosterone-grown strain TUW77 culture revealed estradiol and androstanediol as major end products, with 1-dehydrotestosterone as an intermediate. Elemental composition was determined using Mass-Lynx Mass Spectrometry Software (Waters). *, predicted molecular weight (MW) was calculated using the atom mass of ¹²C (12.0000), ¹⁶O (15.9949), and ¹H (1.0078). (C) ¹³C-NMR spectra of bacterial extracts showing conversion of (2,3,4C-¹³C)testosterone to (2,3,4C-¹³C)estradiol and (2,3,4C-¹³C)androstanediol after 15 d of fermentation. Samples were collected on day 0 (gray) and day 15 (pink), and steroidal metabolites were extracted using ethyl acetate. Chemical shifts (ppm; gray background) for C-2, C-3, and C-4 of ¹³C-labeled steroids were predicted using ChemBioDraw Ultra (version 12.0). Abbreviations: AND, androstanediol; E2, estradiol; T, testosterone.

Fig. 2.

Integrated multi-OMICs analyses reveal the involvement of abe genes and corresponding proteins in anaerobic bacterial estrogenesis. (A) Comparative genomic analysis of Phosphitispora sp. strain TUW77. The paralogous abe genes are mapped on the circular chromosome of strain TUW77, with homologous genes identified in testosterone-catabolizing denitrifying proteobacteria Denitratisoma spp. Homologous open reading frames (colored arrows) between bacterial genomes are connected by gray blocks. (B) Global gene expression profile (RNA-Seq) of strain TUW77 grown anaerobically on testosterone under fermentation conditions. Each spot represents a gene. The FPKM values for genes involved in anaerobic estrogenesis and selected house-keeping genes are provided in SI Appendix, Table S5 and Dataset S2. (C) MS-based proteomics analysis (i) and SDS–PAGE (4 to 20%) (ii) of testosterone-grown strain TUW77. *, each Abe protein band contains multiple paralogs; AbeA (5.2% total abundance) comprises AbeA4 (2.7%), AbeA3 (1.2%), AbeA2 (0.9%), and AbeA1 (0.2%). Relative abundances of individual Abe proteins in the soluble protein fraction and corresponding protein bands are listed in SI Appendix, Table S7. Original gel images are shown in SI Appendix, Fig. S8; data shown are representative of three independent experiments.

Fig. 3.

Phylogenetic analysis of AbeA proteins and characterized cobalamin-dependent methyltransferases. An unrooted maximum likelihood tree was constructed (bootstrap = 1,000). Parentheses indicate uncharacterized methyltransferases from MAGs. SI Appendix, Table S8 provides detailed information about the selected cobalamin-dependent methyltransferases.

Fig. 4.

Microbial enzymes, biochemical mechanisms, and stoichiometry of anaerobic estrogenesis. (A) Proposed mechanisms operated in the androgenic A-ring activation and Abe-catalyzed, cobalamin-mediated C-10 demethylation. (B) TLC analysis showing exclusive estradiol production during anaerobic respiration. Strain TUW77 cells were incubated with and without AQDS for 14 d. The transformation of testosterone into AND occurred only under fermentative conditions. (C) Model of metabolic pathways and stoichiometric equations for testosterone fermentation and AQDS-dependent anaerobic respiration. Abbreviations for microbial metabolites: AcCoA, acetyl coenzyme A; AD, 4-androstene-3,17-dione; ADD, androsta-1,4-diene-3,17-dione; AND, androstanediol; AQDS, anthraquinone-2,6-disulfonate; CoASH, coenzyme A; DT, 1-dehydrotestosterone; E, estrogens (E1 + E2), E1, estrone, estradiol; Fd, ferredoxin; T, testosterone; THF, tetrahydrofolate. Abbreviations for microbial enzymes: ACS, acetyl-CoA synthase; ATPase, adenosine triphosphatase; CODH, carbon monoxide dehydrogenase; CoP, AbeB subunit of 1-dehydrotestosterone methyltransferase; FDH, formate dehydrogenase; FTHFS, formyltetrahydrofolate synthetase; MT1, AbeA subunit of 1-dehydrotestosterone methyltransferase; MT2, MT1, AbeC subunit of 1-dehydrotestosterone methyltransferase; MTHFD, bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase; MTHFR, methylenetetrahydrofolate reductase.