*2.1. Chemicals*

Testosterone (TES), 17β-estradiol (E2), estrone (E1), pyruvate, chloroform, n-hexane, ethyl acetate, sulphuric acid and acetonitrile were purchased from Merck KGaA (Darmstadt, Germany). Androst-4-en-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) were purchased from TCI EUROPE (Boereveldseweg, Belgium). TES-17-acetate (TES-Ac) was purchased from CYMIT QUÍMICA S.L. (Barcelona, Spain). Trans-dehydroandrosterone (DHEA) and pregnenolone (PREG) were purchased from Fluka (Switzerland). Randomly methylated β-cyclodextrin (TRMB-T Randomly Methylated BCD) (CDX) was purchased from Cyclodex (Alachua, USA). Other chemicals and reagents were purchased from Merck KGaA (Darmstadt, Germany).

#### *2.2. Strains and Growth Media*

Bacterial strains and plasmids used in this study are listed in Table S2. *N. tardaugens* NBRC 16725 (strain ARI-1) was purchased from the Leibniz-Institut DSMZ type culture collection. This strain and its mutants were cultured at 30 ◦C in an orbital shaker at 200 rpm. Nutrient broth (NB) (Difco) was used as rich medium to grow this strain. Minimal medium M63 [KH2PO4 (136 g/L), (NH4)2SO4 (20 g/L), FeSO4·7H2O (5 mg/L), pH 7.0] was supplemented with 0.39 mM CaCl2, 1 mM MgSO4 and the appropriate carbon source concentration (we used a carbon equimolar concentration for each substrate tested). Steroids and pyruvate stock solutions were prepared in PBS bu ffer and 70 mM CDX so the final carbon concentration in the culture was 36 mM in 13.33 mM CDX. *Escherichia coli* DH10B, BL21 (DE3) and HB101 strains were grown at 37 ◦C in an orbital shaker at 200 rpm in lysogeny broth (LB) medium [30]. The appropriate antibiotics, i.e., chloramphenicol (34 mg/mL), kanamycin (50 mg/mL) or rifampicin (50 mg/mL) were added when needed.

## *2.3. DNA Manipulation*

Molecular biology and DNA manipulations where performed as described elsewhere [31]. *N. tardaugens* genomic DNA was extracted as described before [29]. Plasmid DNA was purified using High Pure Plasmid Isolation Kit (Roche). DNA fragments where purified with QIAquick PCR Purification Kit (Qiagen) or QIAquick Gel Extraction Kit (Qiagen). *E. coli* cells were transformed using the RbCl method or by electroporation using a Gene Pulser (Bio-Rad) [32]. DNA amplification was performed in a Mastercycler Gradient (Eppendorf) using the oligonucleotides listed in Table S3, which were purchased from Merck KGaA (Darmstadt, Germany). Phusion High-Fidelity DNA Polymerase (New England Biolabs) was used for cloning amplifications and Taq DNA polymerase (Biotools) for screening. All PCR products were checked by agarose gel electrophoresis and those aimed for cloning were confirmed by DNA sequencing by Secugen S.L. (Spain). Digestion of DNA fragments was done using restriction enzymes (New England Biolab) and ligation was performed with Instant Sticky-end Ligase Master Mix (New England Biolabs).

#### *2.4. Gene Expression Analyses*

## 2.4.1. RNA Extraction

Total RNA of *N. tardaugens* cells was extracted from cultures grown in minimal medium with 20 mM CDX and TES or pyruvate as carbon sources. Cells where harvested in mid exponential phase (OD600 0.6) and stored at −80 ◦C. Pellets where thawed and cells were lysed in 400 μL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) containing lysozyme (50 mg/mL) following three freezing–thawing cycles. High Pure Isolation Kit (Roche), followed by DNA-free DNA Removal Kit (Invitrogen) treatment, was used to obtained pure RNA. Purity and concentration were measured in a ND1000 spectrophotometer (Nanodrop Technologies).
