Quence utilizing the Burrows-Wheeler alignment tool (70) and processed with Pilon (71). Sequence reads of genomic DNA had been mapped onto the CEN.PK113-7D genome (63), supplemented with sequences containing the modified SGA1, ACS2, and CAN1 loci, employing the Burrows-Wheeler alignment tool (70). Data were additional processed with Pilon (71), and sequence variations were extracted in the Pilon output file “.modifications.” The uniqueness of sequence variations in strains IMS0482 and IMS0483 was manually confirmed by comparison with strain IMX745 using the Integrative Genomics Viewer (72). Copy quantity variations in strains IMS0482 and IMS0483, relative to strain IMX745, were determined with all the Poisson mixture model-based algorithm Magnolya (37). Growth studies in shake flasks and making use of spot plate assays. For growth research in shake flasks and using spot plates, strains have been pregrown in shake flasks with SM-urea and 20 g liter 1 glucose with lipoic acid or L-carnitine, exactly where proper. For development studies in shake flasks, cells had been washed twice with synthetic medium (61) and transferred to new shake flasks with SM-urea containing 20 g liter 1 glucose and 40 mg liter 1 L-carnitine or 50 ng liter 1 lipoic acid, exactly where indicated. Growth prices had been depending on optical density at 660 nm (OD660) measurements working with a Libra S11 spectrophotometer (Biochrom, Cambridge, Uk). Culture viability was estimated using the FungaLight AMCFDA (acetoxymethyl ester 5-carboxyfluorescein diacetate)/propidium iodide yeast viability kit (Invitrogen, Carlsbad, CA) in addition to a Cell Lab Quanta SC MPL flow cytometer (Beckman Coulter, Woerden, The Netherlands) as described previously (73). For the preparation of spot plates, precultures had been washed once with synthetic medium and diluted in synthetic medium to an OD660 of 0.273 (corresponding to two 106 cells ml 1). Five-microliter samples of a dilution series, containing an estimated two 105, 2 104, and two 103 cells per ml, had been spotted on SM-urea agar plates with 20 g liter 1 glucose and L-carnitine (400 mg liter 1) or lipoic acid (50 ng liter 1) as indicated. Enzyme activity assays. Cell extracts were prepared as described ahead of (eight) from mid-exponentially growing cultures. The growth medium was SM-ammonium with either 20 g liter 1 glucose or two (vol/vol) ethanol as the carbon supply and, where expected, lipoic acid. Activities in cell extracts of carnitine acetyltransferase activity (eight) and glucose-6phosphate dehydrogenase (74) (the latter activity was made use of to verify the high quality of cell extracts) were assayed spectrophotometrically as described previously (8).Methyl 3-fluoroisonicotinate structure Protein concentrations in cell extracts have been determined by the Lowry process (75).1203681-52-0 Purity Nucleotide sequence accession number.PMID:24605203 Raw sequencing information of strains IMX745, IMS0482, and IMS0483 are deposited at the NCBI Sequence Study Archive (http://www.ncbi.nlm.nih.gov/sra) under BioProject identifier (ID) or accession quantity PRJNA313402.SUPPLEMENTAL MATERIALSupplemental material for this article can be discovered at http://mbio.asm.org/ lookup/suppl/doi:ten.1128/mBio.00520-16/-/DCSupplemental. Data Set S1, PDF file, 1 MB. Table S1, DOCX file, 0.04 MB. Table S2, DOCX file, 0.04 MB. Table S3, DOCX file, 0.04 MB.ACKNOWLEDGMENTSWe thank Peter K ter, Annabel Giezekamp, Marlous van Dijk, Henri Duine, Ioannis Papapetridis, and Xavier Hakkaart for assist in strain building and growth research. Pilar de la Torre and Melanie Wijsman are gratefully acknowledged for sequencing plasmids pUDE320 and pUDE321.