Wickerhamomyces ciferrii Auxotroph and Expression Vector for Improved Production of Tetraacetyl Phytosphingosine

Tetraacetyl phytosphingosine (TAPS) is a precursor that has antibacterial and wound-healing properties and is used in the synthesis of ceramides. Wickerhamomyces ciferrii is a non-conventional yeast that has the ability to produce and secrete TAPS extracellularly. However, the genetic engineering tools available for this yeast species are limited. In this study, an auxotrophic strain Y94 of W. ciferrii was developed through random mutation and screening under 1.5 g/L 5-fluoroorotic acid. Additionally, an expression vector was constructed which harbors an uracil selection marker and CEN/ARS origin of replication. Using requisite mutations and expression vectors, we successfully constructed an engineered strain that overexpressed LCB1, LCB2, and SYR2, with the deletion of LCB4, resulting in the highest reported TAPS titer to date (20 g/L) through high cell density fermentation. This study demonstrates the potential of W. ciferrii for TAPS production and highlights the significance of an efficient genetic engineering toolbox in enhancing yields.