title: Combined application of targeted and untargeted proteomics identifies distinct metabolic alterations in the tetraacetylphytosphingosine (TAPS) producing yeast Wickerhamomyces ciferrii.
abstract: The Wickerhamomyces ciferrii strain NRRL Y-1031 F-60-10A is a well-known producer of tetraacetylphytosphingosine (TAPS) and used for the biotechnological production of sphingolipids and ceramides. It was our aim to gain new biological insights into the sphingolipid metabolism by employing a dual platform mass spectrometry strategy. The first step comprised metabolic (15)N-labeling in combination with label-free proteomics using high resolution LTQ Orbitrap mass spectrometry. Then selected reaction monitoring tandem mass spectrometry served for the targeted quantification of sphingoid base biosynthesis enzymes. The non-producer strain NRRL Y-1031-27 served as a reference strain. In total, 1697 proteins were identified, and 123 enzymes of main metabolic pathways were observed as differentially expressed. Major findings were: 1) the likely presence of higher carbon flux in NRRL Y-1031 F-60-10A, resulting in higher availability of pyruvate and acetyl-CoA; 2) indications of oleaginous yeast-like behavior in NRRL Y-1031 F-60-10A; and 3) approx. 2-fold higher abundance of eight sphingolipid biosynthesis enzymes in NRRL Y-1031 F-60-10A. Taken together, in NRRL Y-1031 F-60-10A, the levels of glycolytic enzymes apparently gear carbon flux towards higher acetyl-CoA synthesis rates, while simultaneously reducing protein biosynthesis in the process. Thereby, C2 building blocks for acyl-moieties and downstream sphingoid base acetylation are provided to maintain TAPS production.
  
  
  CONCLUSIONS
  First quantitative proteome study for a phytosphingosine-producing yeast.
authors.affiliation: Dept. of Plant Biochemistry, Ruhr-University Bochum, D-44801 Bochum, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wolff
authors.forename: Frank
authors.initials: F
authors.lastname: ter Veld
authors.forename: Tim
authors.initials: T
authors.lastname: Köhler
authors.forename: Ansgar
authors.initials: A
authors.lastname: Poetsch
mesh_terms: D000105:Acetyl Coenzyme A / Q000096:biosynthesis; D000107:Acetylation; D002518:Ceramides / Q000096:biosynthesis; D005656:Fungal Proteins / Q000096:biosynthesis*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D007553:Isotope Labeling / Q000379:methods; D009587:Nitrogen Isotopes / Q000494:pharmacology; D020543:Proteome / Q000096:biosynthesis*; D040901:Proteomics / Q000379:methods*; D004718:Saccharomycetales / Q000378:metabolism*; D013107:Sphingolipids / Q000096:biosynthesis*
chemical_list: D002518:Ceramides; D005656:Fungal Proteins; D009587:Nitrogen Isotopes; D020543:Proteome; D013107:Sphingolipids; D000105:Acetyl Coenzyme A