Additional file 1 of Genetic inactivation of the Carnitine/Acetyl-Carnitine mitochondrial carrier of Yarrowia lipolytica leads to enhanced odd-chain fatty acid production
Creators: name: Messina, Eugenia
name: de Souza, Camilla Pires
name: Cappella, Claudia
name: Barile, Simona Nicole
name: Scarcia, Pasquale
name: Pisano, Isabella
name: Palmieri, Luigi
name: Nicaud, Jean-Marc
name: Agrimi, Gennaro
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified

description: Additional Tables: Additional Table 1. Identity Matrix of CAC mitochondrial carriers. Percentage of amino acidic identity among Carnitine/Acetyl-Carnitine mitochondrial carriers of Saccharomyces cerevisiae (ScCrc1), Aspergillus nidulans (AnCac), Homo sapiens (HsCact) and the two putative CAC transporters of Yarrowia lipolytica (YlCrc1 and YALI0A20988p). The identity matrix was created by using the web tool “SIAS Sequence Identity And Similarity”. Additional Table 2. Identity Matrix of CAT enzymes of S. cerevisiae and Y. lipolytica. Percentage of amino acids identity among carnitine acetylcarnitine transferases (CATs) of S. cerevisiae (Cat2, Yat1 and Yat2) and the two putative CAT enzymes of Y. lipolytica (YALI0B10304p and YALI0F21197p). The identity matrix was created by using the web tool “SIAS Sequence Identity And Similarity”. Additional Table 3. Lipid profile of Y. lipolytica strains. Lipid profile (% of total lipids) of wild type (WT), Ylcrc1?, pTEF-RePCT and pTEF-RePCT Ylcrc1Δ strains on D2P0.5A1 and D3P0.5 lipogenic media. The strains were cultivated on nitrogen-limited media (1.5 g/L NH4Cl) for 120 hours at 28°C. Averages and standard errors were obtained from at least three replicate experiments. Additional Table 4. Primers used in this study. Additional Figures: Additional Figure 1. Alignment of amino acid sequences of CAC mitochondrial carriers. Alignment of CAC transporters of H. sapiens (HsCact), A. nidulans (AnCac), S. cerevisiae (ScCrc1) and its closest homologs in Y. lipolytica, YlCrc1 and YALI0A20988p. The three signature motifs of the mitochondrial carrier family of transporters, PX[DE]XX[RK]X[RK] are underlined; in black box the distinct motif RXXPANAAXF of CAC transporters and in red the identical amino acids. Sequences were aligned with NPS@: Network Protein Sequence Analysis. Additional Figure 2. Comparison of carbon source consumption. Comparison of carbon source consumption in wild type (blue) and strain with deletion of YlCRC1 (green). A) Glucose, B) acetate, and C) ethanol. For ethanol a correction based on the evaporation rate was applied. Averages and standard errors were obtained from two replicate experiments. Additional Figure 3. Subcellular localization of YlCrc1 mitochondrial carrier. Subcellular localization of YlCrc1 fusion protein after expression in Y. lipolytica CRC1 knock-out strain. MitoTracker Green was used to locate mitochondria in the cells (MitoTracker panel), and phase contrast microscopy to monitor the integrity of the cells. The same cells were photographed first with a RedStar2 filter set and then with the MitoTracker filter set. Identical fields are presented. Additional Figure 4. Alignment of amino acid sequences of CAT enzymes of S. cerevisiae and Y. lipolytica. Multiple alignment of the five amino acid sequences of the CATs of S. cerevisiae and the putative CATs of Y. lipolytica. Cat2 and YALI0B10340p contain a mitochondrial targeting sequence (MTS) at the N-terminus (yellow) and a functional variant of the peroxisomal targeting signal type 1 (PTS-1) AKL at C-terminal (green). Cat2 and YALI0B10340p display moreover a second in-frame ATG codon, placed after the MTS (M in red). All five sequences show the LPXLPXPXL motif of choline and carnitine acetyltransferases (black box). Sequences were aligned with NPS@: Network Protein Sequence Analysis. Additional Figure 5. Oleate-response element (ORE). Comparison of the oleate-response element (in bold) of S. cerevisiae (Sc_crc1) and that of Y. lipolytica (Yl_crc1 and YALIA20988) found based on 5’-CGGN3TNAN9-12CCG-3’ sequence. In grey, the start of the CDS with the ATG initiation codon underlined. Additional Figure 6. 10.1186/s12934-023-02137-8 Real-time qPCR of mRNAs expression of YlCRC1, YALI0A20988, YlCAT2 and YlYAT1 on lipogenic media. Expression of YlCRC1, YALI0A20988g, YlCAT2 and YlYAT1 in wild-type cells grown on D2P.05A1 (light blue) and D3P0.5 (green) media after 15 hours (exponential phase) (A) and 72 hours (stationary phase) (B). mRNA levels were quantified by qPCR, and the mRNA of YlCRC1 gene, grown on glucose supplemented medium at 15 hours, was used as calibrator. The quantification of relative gene expression was calculated according to the comparative method (2-ΔΔCCt). Averages and standard errors were obtained from at least three independent experiments.