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Shaking for 96 h at 28 . For the feeding experiment with OA, the 4-ml cultures had been grown within the presence or absence of 0.5 mM orsellinic acid (Alfa Aesar, Heysham, England) in dimethyl sulfoxide (DMSO).February 2021 Volume 87 Issue 3 e01510-20 aem.asm.orgReyes-Fern dez et al.Applied and Environmental MicrobiologyMetabolite extraction NF-κB Agonist drug evaluation and structure elucidation. Pelleted cells of 30-ml liquid cultures were transferred to 50-ml glass beakers and stirred with 20 ml acetone for 1 h. The extracts have been filtered into 100-ml round-bottom flasks, and the pellets have been washed using a further 10 ml acetone before getting concentrated to dryness within a rotary evaporator. Dry extracts have been dissolved in two ml methanol and transferred to 4-ml glass vials before becoming concentrated to dryness inside a speed β-lactam Chemical Accession vacuum (Speed VacConcentrator, Eppendorf, Hamburg) for two h at 28 . Extracts were dissolved in 1 ml of methanol (highpressure liquid chromatography [HPLC] grade) and centrifuged (16,000 rpm, 20 min) to make sure that no particles had been injected in to the HPLC instrument. Ultimately, one hundred m l on the supernatant was transferred to HPLC plastic vials and subjected to analysis. For large-scale cultures (.1 liter), the entire volume was placed into a 2-liter flask with 30 g of Amberlite XAD-16 beads (Sigma-Aldrich) and agitated at 200 rpm for 2 h at four . Afterward, the Amberlite beads have been harvested and stored at 220 for additional analysis. HPLC-MS evaluation was performed using a Dionex UltiMate 3000 technique coupled to a Bruker AmaZon X mass spectrometer and an Acquity UPLC BEH C18 1.7 m m reverse phase (RP) column (Waters). Bioinformatic DNA sequence analysis. All DNA and protein sequences have been retrieved in the National Center for Biotechnology Data (NCBI) database (www.ncbi.nlm.nih.gov). Homology searches were performed applying BLASTp with default settings (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Data availability. The following genes had been applied in this study: pks5 (UMAG_04095) (GenBank accession quantity XM_011392301), orf1 (UMAG_04096) (XM_011392302), pks4 (UMAG_04097) (XM_011392303), orf2 (UMAG_04098) (XM_011392304), mtf2 (UMAG_11110) (XM_011392495), orf3 (UMAG_04100) (XM_ 011392305), mtf1 (UMAG_04101) (XM_011392306), aox1 (UMAG_11111) (XM_011392496), vbs1 (UMAG_ 11112) XM_011392497), orf4 (UMAG_04104) (XM_011392307), pks3 (UMAG_04105) (XM_011392308), omt1 (UMAG_04106) (XM_011392309), pmo1 (UMAG_04107) (XM_011392310), orf5 (UMAG_12253) (XM_ 011392513), cyp4 (UMAG_04109) (XM_011392311), deh1 (UMAG_11113) (XM_011392498), and UMAG_05798 (XP_011392172.1. Software program. Phylogenetic evaluation of the KS domains of fungal PKSs was performed by utilizing the system MEGAX version ten.1.8. Figure 1 was drawn applying the plan BioRender.SUPPLEMENTAL MATERIAL Supplemental material is readily available on-line only. SUPPLEMENTAL FILE 1, PDF file, 4.8 MB. ACKNOWLEDGMENTS We’re grateful to Zakaria Cheikh, Victoria Challinor, and Lisa Rosenbecker, who supplied insight and knowledge that assisted this study. We also thank Marc Strickert for his guidance within the identification of possible U. maydis gene clusters, Marino Moretti for delivering technical tips and the plasmid pMM69, and Kenan Bozh for aid together with the dehydratase analysis. Perform within the B ker lab was supported by DFG-funded grant SFB987 and by the LOEWE Zentrum SYNMIKRO funded by the state of Hesse. Function inside the Bode lab was supported by the LOEWE Schwerpunkt MegaSyn and the LOEWE Zentrum TBG, both funded by the state of Hesse.
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