Abstract
Wickerhamomyces ciferrii is a microorganism characterized by the production and secretion of large amounts of acetylated sphingoid bases, in particular tetraacetyl phytosphingosine. Here, we present the 15.90-Mbp draft genome sequence of W. ciferrii NRRL Y-1031 F-60-10 generated by pyrosequencing and de novo assembly. The draft genome sequence comprising 364 contigs in 150 scaffolds was annotated and covered 6,702 protein-coding sequences. This information will contribute to the metabolic engineering of this yeast to improve the yield and spectrum of acetylated sphingoid bases in biotechnological production.
GENOME ANNOUNCEMENT
The ascomycetous yeast Wickerhamomyces ciferrii (synonym Pichia ciferrii) is a natural producer of sphingolipid derivatives. Acetylated sphingoid bases are excreted into culture media from which they can be recovered and chemically converted into ceramides (11). In mammals, ceramides serve important functions related to formation and maintenance of the skin's epidermal permeability barrier, protecting skin from desiccation and preventing harmful external factors from penetrating the skin (8, 9). They have important antimicrobial and anti-inflammatory functions accounting for effective barrier homeostasis (3, 13) and induce keratinocyte differentiation (12), thereby promoting efficient stratum corneum barrier formation. The efficacy of ceramides has been demonstrated to depend on proper stereochemistry. It is noteworthy that sphingoid bases produced by W. ciferrii feature the same stereochemistry as sphingoid bases found in human skin, consequently making these sphingoid bases skin identical. Therefore, these sphingolipids are valuable active ingredients used for pharmaceutical and cosmetic applications (5, 7). W. ciferrii is already used industrially for fermentative production of tetraacetyl phytosphingosine (TAPS), and metabolic engineering methods are available (2, 4, 15, 16).
The draft genome sequence of W. ciferrii NRRL Y-1031 F-60-10 was established by whole-genome shotgun and 3-kb paired-end sequencing according to the manufacturer's manuals (Roche/454). This approach generated 534 Mbp of genomic information and 2,628,268 sequencing reads, including 580,095 paired-end reads. Sequencing data were assembled with the GS De Novo Assembler 2.6 to 673 contigs with a total length of 16.04 Mbp (33-fold coverage). A total of 364 contigs were ordered into 150 scaffolds with a total length of 15.90 Mbp. The average G+C content is 30.4%, which is relatively low compared to published Saccharomycotina yeast genomes (6).
Annotation of the draft genome was performed with RAPYD (14). The embedded gene prediction tool Augustus (17) and a manual verification identified 6,702 protein-coding genes that comprise 5,688 single-exon genes and 1,014 multiexon genes. Coding regions have an average length of 1,456 bp and cover 61.36% of the genome. Functional annotations assigned 4,253 descriptions, 1,719 EC numbers, and 707 gene names.
Metabolic pathway reconstructions confirmed the presence of genes involved in glycolysis, gluconeogenesis, tricarboxylic acid (TCA) cycle, fatty acid and serine biosynthesis, and sphingolipid metabolism. The existence of sphingolipid metabolism genes is in accordance with recent studies (4, 15). Due to the ability of W. ciferrii NRRL Y-1031 F-60-10 to secrete acetylated sphingoid bases, protein sequences were screened for Rsb1p-like sequences using BLASTp (1). Rsb1p (GI:6324623) encodes a transporter of sphingoid long-chain bases in Saccharomyces cerevisiae S288c (10). As a result, two proteins encoded by adjacently located genes were identified (BN7_1410 and BN7_1411), separated by a 1,801-bp intergenic region. Both proteins show an identity of about 40% to S. cerevisiae Rsb1p. These Rsb1p-like sequences might contribute to the export of sphingolipid derivatives.
Beyond the fundamental knowledge that has been established for W. ciferrii NRRL Y-1031 F-60-10, the draft genome presents a basis for the identification of target genes contributing to further improvement of sphingoid base production by metabolic engineering as presented in recent studies (4, 15).
Nucleotide sequence accession numbers.
The sequences have been deposited in EMBL-EBI nucleotide database and assigned accession numbers CAIF01000001 to CAIF01000364.
ACKNOWLEDGMENTS
This work was funded by Evonik Industries AG and the Federal Ministry of Education and Research (BMBF), funding number 0315191. J.S. and K.B. acknowledge the CLIB Graduate Cluster Industrial Biotechnology. We thank the BRF system administrators for technical support.
A.G., A.P., S.J., and J.B. received financial support from the GenoMik-Transfer program (grants 0315599A and 0315599B) of the BMBF.
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