droxy-9,10-secoandrosta-1,3,five(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-1,3,5(ten)7-tetraene-6,17-dione).Microorganisms 2021, 9,four ofThe ambitions of this study had been initial to further investigate 9-hydroxylation in strain Chol11 by supplying DHSATD as a substrate. As this resulted within the identification of a second side reaction leading to an unprecedented side solution, the presence of both pathway variants inside the soil, as well as effects of your known side product THADD and also the newly discovered side item, were analyzed. two. Material and Approaches 2.1. Cultivation of Bacteria Strains of Pseudomonas stutzeri Chol1 (DSM 103613) [7] and Sphingobium sp. strain Chol11 (DSM 110934) [21] were 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] had 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 pBBR1MCS5 [33] have been 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 had 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 1.5 (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. 2.two. Growth Experiments and Co-Cultures Growth experiments and co-cultures were performed in three mL medium in 10 mL test tubes at 30 C and orbital shaking (Minitron or Ecotron, Infors HT, Einsbach, Germany). Precultures had been grown with the respective carbon GCN5/PCAF Activator manufacturer supply for about 17 h and added to major cultures without the need of prior washing. DHSATD (XI in Figure 1) was added in concentrations equaling the two-fold concentration developed in cultures of P. stutzeri Chol1 pBBR1MCS5::hsh2 cultivated with 1 mM cholate. MDTETD (XIII) was added in concentrations equaling the ten-fold concentration developed in co-cultures of P. stutzeri Chol1 pBBR1MCS-5::hsh2 and Sphingobium sp. strain Chol11 sclA cultivated with 1 mM cholate. Development was tracked by measuring the optical density at 600 nm (OD600 ) (Camspec M107, Spectronic Camspec, UK). Samples for HPLC-MS measurements were 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 six h. Most important cultures containing the same carbon CysLT2 Antagonist medchemexpress source were seeded with all the preculture to OD600 = 0.015 and incubated at 30 C with orbital shaking at 200 rpm for about 16 h. In the exponential growth phase, cells have been harvested by centrifugation at 8000g and 4 C for 8 min. Cells have been washed and resuspended in MB medium without having a carbon supply. Cell suspensions had been diluted to defined OD600 values. Samples for HPLC-MS measurements had been withdrawn instantaneously right after adding DHSATD (concentration as described) or cholate