droxy-9,10-secoandrosta-1,3,5(10),6-tetraene-9,17-dione), XII: THADD (1,two,12-Trihydroxy-androsta-4,6-triene3,17-dione), XIII: MDTETD (4-Methyl-3-deoxy-1,9,12-trihydroxyestra-1,three,five(ten)7-tetraene-6,17-dione).Microorganisms 2021, 9,four ofThe objectives of this study had been 1st to additional investigate 9-hydroxylation in strain Chol11 by offering DHSATD as a substrate. As this resulted within the identification of a second side reaction major to an unprecedented side item, the presence of each pathway variants within the soil, at the same time as effects on the known side product THADD along with the newly discovered side solution, have been analyzed. two. Material and Procedures two.1. Cultivation of Bacteria Strains of Pseudomonas stutzeri Chol1 (DSM 103613) [7] and Sphingobium sp. strain Chol11 (DSM 110934) [21] had been grown within the HEPES buffered mineral medium MB as described previously [21,29]. P. stutzeri Chol1, Sphingobium sp. strain Chol11 wt and nov2c349 had been grown with 1 mM cholate as carbon source, P. stutzeri Chol1 pBBR1MCS5::hsh2 [22] and P. stutzeri Chol1 kstD1 stdA1 pBBR1MCS-5::hsh2 [11] have been grown with 12 mM succinate and Sphingobium sp. strain Chol11 sclA [25] was grown with 15 mM glucose. Escherichia coli strains and most other strains containing pDM4 [32] or IL-6 Inhibitor manufacturer pBBR1MCS5 [33] were cultivated in lysogeny broth medium (LB) [34] with respective antibiotics at 30 C. For cultivating E. coli ST18 [35], 50 mL-1 5-aminolevulinic acid had been added. Strains containing pDM4 have been cultivated with 30 or 90 mL-1 chloramphenicol, and strains containing pBBR1MCS-5::hsh2 were cultivated with 20 mL-1 gentamicin. Strains have been maintained on agar plates, prepared in the aforementioned media with 1.five (w/v) Bacto agar (BD, Sparks, USA) and with either cholate for strains Chol1 and Chol11, glucose for mutant strains of strain Chol11, or succinate and gentamicin for strain Chol1 pBBR1MCS-5::hsh2 and strain Chol1 kstD1 stdA1 pBBR1MCS-5::hsh2. two.two. Development Experiments and Co-D4 Receptor Agonist review cultures Development experiments and co-cultures have been performed in 3 mL medium in ten mL test tubes at 30 C and orbital shaking (Minitron or Ecotron, Infors HT, Einsbach, Germany). Precultures were grown with the respective carbon source for about 17 h and added to principal cultures devoid of earlier washing. DHSATD (XI in Figure 1) was added in concentrations equaling the two-fold concentration produced in cultures of P. stutzeri Chol1 pBBR1MCS5::hsh2 cultivated with 1 mM cholate. MDTETD (XIII) was added in concentrations equaling the ten-fold concentration produced in co-cultures of P. stutzeri Chol1 pBBR1MCS-5::hsh2 and Sphingobium sp. strain Chol11 sclA cultivated with 1 mM cholate. Growth was tracked by measuring the optical density at 600 nm (OD600 ) (Camspec M107, Spectronic Camspec, UK). Samples for HPLC-MS measurements have been withdrawn at defined time points. 2.3. Cell Suspension Experiments For cell suspensions of Sphingobium sp. strain Chol11, a preculture with 1 mM cholate or 15 mM glucose was incubated for 6 h. Most important cultures containing the same carbon source had been seeded using the preculture to OD600 = 0.015 and incubated at 30 C with orbital shaking at 200 rpm for about 16 h. Inside the exponential development phase, cells have been harvested by centrifugation at 8000g and four C for eight min. Cells have been washed and resuspended in MB medium with no a carbon supply. Cell suspensions were diluted to defined OD600 values. Samples for HPLC-MS measurements have been withdrawn instantaneously immediately after adding DHSATD (concentration as described) or cholate
