Combined application of targeted and untargeted proteomics identifies distinct metabolic alterations in the tetraacetylphytosphingosine (TAPS) producing yeast Wickerhamomyces ciferrii

• Published in: Journal of proteomics Vol. 82; pp. 274 - 287
• Main Authors: Wolff, Daniel, ter Veld, Frank, Köhler, Tim, Poetsch, Ansgar
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 26.04.2013
• Subjects: acetyl coenzyme A; Acetyl Coenzyme A - biosynthesis; Acetylation; biochemical pathways; biosynthesis; carbon; ceramides; Ceramides - biosynthesis; Comparative proteomics; enzymes; Fungal Proteins - biosynthesis; gene expression regulation; Gene Expression Regulation, Fungal - physiology; glycolysis; Isotope Labeling - methods; monitoring; Nitrogen Isotopes - pharmacology; Phytosphingosine; protein synthesis; proteins; proteome; Proteome - biosynthesis; proteomics; Proteomics - methods; pyruvic acid; Saccharomycetales - metabolism; Sphingoid base; Sphingolipid; Sphingolipids - biosynthesis; tandem mass spectrometry; Wickerhamomyces ciferrii; Yeast; yeasts; AQUA; Yeast; Sphingolipid; LCB; TriASa; SRM; Sphingoid base; RP-HPLC; SC; Phytosphingosine; AU; FDR; UPLC; SPT; TAPS; XCorr; Comparative proteomics
• ISSN: 1874-3919; 1876-7737
• DOI: 10.1016/j.jprot.2013.03.002

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 15N-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. First quantitative proteome study for a phytosphingosine-producing yeast. [Display omitted] •Combined targeted/untargeted approach:•allowed comprehensive coverage of central metabolic enzymes•resulted in complete coverage of relevant phytosphingosine biosynthesis pathway•revealed oleaginous yeast-like behavior for the general physiology of producing strain