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

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Title
Integrative transformation system for the metabolic engineering of the sphingoid base-producing yeast Pichia ciferrii
Author(s)
Jung Hoon BaeJung Hoon Sohn; C S Park; J S Rhee; Eui Sung Choi
Bibliographic Citation
Applied and Environmental Microbiology, vol. 69, no. 2, pp. 812-819
Publication Year
2003
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
Publisher
Amer Soc Microb
DOI
http://dx.doi.org/10.1128/AEM.69.2.812-819.2003
Type
Article
Appears in Collections:
Division of Biomaterials Research > Synthetic Biology and Bioengineering Research Center > 1. Journal Articles
Division of Bio Technology Innovation > BioProcess Engineering Center > 1. Journal Articles
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