Production of tetraacetyl phytosphingosine (TAPS) in Wickerhamomyces ciferrii is catalyzed by acetyltransferases Sli1p and Atf2p

Abstract

Wickerhamomyces ciferrii secretes tetraacetyl phytosphingosine (TAPS), and in this study, the catalyzing acetyltransferases were identified using mass spectrometry-based proteomics. The proteome of wild-type strain NRRL Y-1031 served as control and was compared to the tetraacetyl phytosphingosine defective mating type NRRL Y-1031-27. Acetylation of phytosphingosine in W. ciferrii is catalyzed by acetyltransferases Sli1p and Atf2p, encoded by genes similar to Saccharomyces cerevisiae YGR212W and YGR177C, respectively. Ablation of SLI1 resulted in an almost complete loss of tri- and tetraacetyl phytosphingosines, whereas the loss ATF2 resulted in an 15-fold increase in triacetyl phytosphingosine. Most likely, it is the concerted action of these two acetyltransferases that yields tetraacetyl phytosphingosine, in which Sli1p catalyzes initial O- and N-acetylation, producing triacetyl phytosphingosine. Finally, Atf2p catalyzes final O-acetylation to yield tetraacetyl phytosphingosine. The current study demonstrates that mass spectrometry-based proteomics can be employed to identify key steps in ill-explored metabolite biosynthesis pathways of nonconventional microorganisms. Furthermore, the identification of phytosphingosine as substrate for alcohol acetyltransferase Atf2p broadens the known substrate range of this enzyme. This interesting property of Atf2p may be exploited to enhance the secretion of heterologous compounds.

Differences in the Fatty Acid Profile, Morphology, and Tetraacetylphytosphingosine-Forming Capability Between Wild-Type and Mutant Wickerhamomyces ciferrii

“…Sphinganine is hydroxylated into phytosphingosine by sphinganine C-4-hydroxylase (encoded by the SYR2 gene). Last, phytosphingosine is acetylated, meaning that TAPS is formed at this point by sphingoid base N/O-acetyltransferases (encoded by the genes Slli1 and Atf2 ) ( Börgel et al, 2012 ; Schorsch et al, 2012 ; Ter Veld et al, 2013 ).…”

EngineeringYarrowia lipolyticaforde novoproduction of tetraacetyl phytosphingosine

“…Several studies have increased TAPS production from W. ciferrii (from 21 mg TAPS g DCW À1 up to 199 mg TAPS g DCW À1 ) (Casey et al 1997;Bae et al 2003;Schorsch et al 2012). The unique acetyl transferase activities of Sli1p and Atf2p in W. ciferrii have been identified as one of the main reasons for the efficient production of acetylated sphingoid bases in W. ciferrii (Wickerham and Stodola 1960;ter Veld et al 2013). The production of di-or tri-acetylated phytosphingosine in other yeasts has been attempted through introduction of W. ciferrii Sli1p or Atf2p (ter Veld et al 2013).…”