Integrative Transformation System for the Metabolic Engineering of the Sphingoid Base-Producing Yeast Pichia ciferrii

Abstract

We have developed an integrative transformation system for metabolic engineering of the tetraacetyl phytosphingosine (TAPS)-secreting yeast Pichia ciferrii. The system uses (i) a mutagenized ribosomal protein L41 gene of P. ciferrii as a dominant selection marker that confer resistance to the antibiotic cycloheximide and (ii) a ribosomal DNA (rDNA) fragment of P. ciferrii as a target for multicopy gene integration into the chromosome. A locus within the nontranscribed region located between 5S and 26S rDNAs was selected as the integration site. A maximum frequency of integrative transformation of approximately 1,350 transformants/g of DNA was observed. To improve the de novo synthesis of sphingolipid, the LCB2 gene, encoding a subunit of serine palmitoyltransferase, which catalyzes the first committed step of sphingolipid synthesis, was cloned from P. ciferrii and overexpressed under the control of the P. ciferrii glyceraldehyde-3-phosphate dehydrogenase promoter. After transformation of an LCB2 gene expression cassette, several transformants that contained approximately five to seven copies of transforming DNA in the chromosome and exhibited about 50-fold increase in LCB2 mRNA relative to the wild type were identified. These transformants were observed to produce approximately two times more TAPS than the wild type.Sphingolipids are integral membrane components found in mammalian cells. They are also distributed widely in plants and microbes (14). Sphingolipids constitute a diverse group of complex membrane lipids, all containing a long-chain amino acid alcohol, usually of 18 carbons, connected by an amide linkage to a fatty acid to form ceramide. Sphingolipids have structural functions in maintaining cell membrane integrity, and they act as anchors for proteins. Metabolites of sphingolipids, such as ceramide, sphingosine, and sphingosine 1-phosphate, are involved as bioeffector molecules and second messengers in cell growth, differentiation, cell senescence, apoptosis, and stress responses (for reviews, see references 9, 29, and 20). Ceramide is a major end product of differentiation through the keratinization process in the mammalian epidermis and is secreted into the extracellular space to form a mantle surrounding individual keratinized cells (19). Extracellular ceramide is a major part of the permeability barrier and the water reservoir of the skin (11). Therefore, ceramide has attracted great attention as a specialty ingredient for moisture retention and protection of the skin in the cosmetics industry. Extraction of ceramides from natural sources, however, is difficult, and in some cases the resulting product is not acceptable for cosmetic use. Chemically synthesized ceramides are also available as a mixture of stereoisomers that must be separated prior to use. Biological production of sphingoid base, such as sphinganine and phytosphingosine, can provide the base materials for conversion to various ceramides. This sphingoid base can be obtained from several yeasts and easily converted to ceramides by N a...

Metabolic engineering of the non-conventional yeast Pichia ciferrii for production of rare sphingoid bases

“…Transformation of S. cerevisiae and P. ciferrii strains was performed according to published protocols (Bae et al, 2003;Gietz and Woods, 2002).…”

“…The sequence of the glyceraldehyde 3-phosphate dehydrogenase encoding gene had been reported previously (Bae et al, 2003).…”

“…Besides the gene encoding one subunit of serine C-palmitoyItransferase (LCB2) which has been isolated and functionally characterized previously (Bae et al, 2003) no other P. ciferrii genes encoding enzymes of sphingolipid biosynthesis were known at the onset of this study. As described above sphinganine is the precursor of phytosphingosine and phytosphingosine-derived sphingolipids, but also precursor of a second branch of sphingolipid biosynthesis yielding sphingolipids containing desaturated sphingoid bases.…”

Cloning and functional characterization of the SUR2/SYR2 gene encoding sphinganine hydroxylase in Pichia ciferrii

“…These secreted bases consist mostly of tetraacetyl phytosphingosine (TAPS), which is a precursor of sphingolipid (Barenholz et al , 1973). Recently, we constructed TAPS‐overproducing strains by metabolic engineering of P. ciferrii (Bae et al , 2003). In order to metabolically engineer P. ciferrii to produce various sphingoid bases and to investigate the function of the hydroxyl groups of phytosphingosine, we cloned the P. ciferrii gene homologue of the S. cerevisiae SUR2 ( ScSUR2 ) gene and characterized the production of sphingoid bases in an S. cerevisiae sur2 ‐null mutant complemented by expression of the P. ciferrii SUR2 ( PcSUR2 ) gene.…”

Lipid biotechnology: Industrially relevant production processes

“…Finally, the sphingosine is acetylated and excreted by Pichia ciferri. A transformation system for the yeast has been developed, which allows for the metabolic engineering of the tetraacetyl-phytosphingosine synthesis and excretion pathway [101]. The cloning of 11 genes of the P. ciferrii phytosphingosine pathway was recently published [102].…”