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Title 

Integrative transformation system for the metabolic engineering of the sphingoid base-producing yeast Pichia ciferrii

Authors 

Jung Hoon BaeJung Hoon SohnC S ParkJ S RheeEui Sung Choi

Publisher 

American Society for Microbiology

Issue Date 

2003

Citation 

Applied and Environmental Microbiology, vol. 69, no. 2, pp. 812-819

Keywords 

yeastmetabolic engineeringcell transformationpichiagenetic engineeringtransformation, geneticpichia 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/μg 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.

ISSN 

0099-2240

Link 

http://dx.doi.org/10.1128/AEM.69.2.812-819.2003

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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