Integrative transformation system for the metabolic engineering of the sphingoid base-producing yeast Pichia ciferrii.
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/ microg 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.
Bae
Jung-Hoon
JH
Laboratory of Microbial Functions, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-333, Korea.
Sohn
Jung-Hoon
JH
Park
Chang-Seo
CS
Rhee
Joon-Shick
JS
Choi
Eui-Sung
ES
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D020459:Genes, rRNA; D005818:Genetic Engineering / Q000379:methods*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D051102:Serine C-Palmitoyltransferase; D013107:Sphingolipids / Q000378:metabolism*; D014170:Transformation, Genetic
D012269:Ribosomal Proteins; D013107:Sphingolipids; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D000217:Acyltransferases; D051102:Serine C-Palmitoyltransferase