Patent ID: 8372595

Claim:
A method to obtain a Pichia ciferrii strain that produces at least 0.5 mg per g cell dry weight of sphingosine and triacetylated sphingosine comprising obtaining a syringomycinE-resistant Pichia ciferrii strain defective in dihydrosphingosine C-4 hydroxylase; inactivating or deleting a genomic sphingolipid Δ8-desaturase (8DES) gene encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6 and/or inactivating a genomic alkaline ceramidase (YXC1) gene encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:8 in the syringomycinE-resistant Pichia ciferrii strain defective in dihydrosphingosine C-4 hydroxylase; increasing expression of i) a polynucleotide encoding an enzyme having dihydroceramide-Δ4-desaturase activity and comprising the amino acid sequence of SEQ ID NO:17 or an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO:17, ii) a polynucleotide encoding an enzyme having ceramidase activity, said ceramidase hydrolyzing sphingosine and triacetylated sphingosine, and comprising the amino acid sequence of SEQ ID NO:15 or an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO:15, and iii) a polynucleotide encoding an enzyme having ceramide synthase activity and comprising the amino acid sequence of SEQ ID NO:2, 4, 9, 10, 12, or 14 or an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO:2, 4, 9, or 10, wherein increasing expression is by transformation of the Pichia ciferrii strain with the polynucleotides and optionally integrating one or several copies of the polynucleotides into the chromosome of the Pichia ciferrii strain; and isolating a Pichia ciferrii strain that produces at least 0.5 mg per g cell dry weight of sphingosine and triacetylated sphingosine.