droxy-9,10-secoandrosta-1,3,5(10),6-tetraene-9,17-dione), XII: THADD (1,2,12-Trihydroxy-androsta-4,6-triene3,17-dione), XIII: MDTETD (4-Methyl-3-deoxy-1,9,12-trihydroxyestra-1,three,five(10)7-tetraene-6,17-dione).Microorganisms 2021, 9,4 ofThe ambitions of this study had

droxy-9,10-secoandrosta-1,3,5(10),6-tetraene-9,17-dione), XII: THADD (1,2,12-Trihydroxy-androsta-4,6-triene3,17-dione), XIII: MDTETD (4-Methyl-3-deoxy-1,9,12-trihydroxyestra-1,three,five(10)7-tetraene-6,17-dione).Microorganisms 2021, 9,4 ofThe ambitions of this study had been very first to additional investigate 9-hydroxylation in COX Activator manufacturer strain Chol11 by offering DHSATD as a substrate. As this resulted in the identification of a second side reaction leading to an unprecedented side product, the presence of both pathway variants in the soil, too as effects of your identified side item THADD plus the newly found side product, were analyzed. 2. Material and Methods two.1. Cultivation of Bacteria Strains of Pseudomonas stutzeri Chol1 (DSM 103613) [7] and Sphingobium sp. strain Chol11 (DSM 110934) [21] have been grown inside 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] were 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] had 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 were added. Strains containing pDM4 were cultivated with 30 or 90 mL-1 chloramphenicol, and strains containing pBBR1MCS-5::hsh2 had been cultivated with 20 mL-1 gentamicin. Strains had been maintained on agar plates, prepared from 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.2. Growth Experiments and Co-Cultures Development 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 have been grown with the respective carbon supply for about 17 h and added to main cultures with out previous washing. DHSATD (XI in Figure 1) was added in concentrations equaling the two-fold concentration created 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. 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 IL-8 Antagonist Species defined time points. 2.three. 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. Key cultures containing the same carbon source had been seeded with all the preculture to OD600 = 0.015 and incubated at 30 C with orbital shaking at 200 rpm for about 16 h. Inside the exponential growth phase, cells were harvested by centrifugation at 8000g and four C for eight min. Cells were washed and resuspended in MB medium without having a carbon supply. Cell suspensions have been diluted to defined OD600 values. Samples for HPLC-MS measurements have been withdrawn immediately following adding DHSATD (concentration as described) or cholate