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Knockout of the DNA ligase IV homolog gene in the sphingoid base producing yeast Pichia ciferrii significantly increases gene targeting efficiency
- Source :
- Current Genetics. 55:381-389
- Publication Year :
- 2009
- Publisher :
- Springer Science and Business Media LLC, 2009.
-
Abstract
- The yeast Pichia ciferrii produces large quantities of the sphingoid base tetraacetyl phytosphingosine (TAPS) and is an interesting platform organism for the biotechnological production of sphingolipids and ceramides. Ceramides have attracted great attention as a specialty ingredient for moisture retention and protection of the skin in the cosmetics industry. First attempts have been started to metabolically engineer P. ciferrii for improved production of TAPS and other sphingoid bases. However, rational metabolic engineering of P. ciferrii is difficult due to a low gene targeting efficiency. In eukaryotes, two major pathways coexist, which are responsible for genomic DNA integration, homologous recombination (HR) and non-homologous end joining (NHEJ). Integration via HR is targeted, while NHEJ involves ectopic (non-targeted) integration depending on a ligation step mediated by DNA ligase IV (Lig4). Here, we demonstrate a dramatical increase in gene targeting efficiency in a P. ciferrii lig4 knockout strain, deficient in NHEJ. Furthermore, a quick and easy to use freeze-thaw method was developed to transform P. ciferrii with high efficiency. Owing to the ability of targeting genomic DNA integration our results pave the way for further genetic and metabolic engineering approaches with P. ciferrii by means of knocking out or overexpressing predestinated genes.
- Subjects :
- DNA Ligases
Genes, Fungal
Genetic Vectors
Biology
LIG4
Pichia
Metabolic engineering
DNA Ligase ATP
Transformation, Genetic
Genetics
DNA, Fungal
Gene
Recombination, Genetic
chemistry.chemical_classification
Sphingolipids
DNA ligase
Models, Genetic
Fungal genetics
Gene targeting
Sequence Analysis, DNA
General Medicine
Cell biology
Non-homologous end joining
chemistry
Gene Targeting
Homologous recombination
Gene Deletion
Plasmids
Subjects
Details
- ISSN :
- 14320983 and 01728083
- Volume :
- 55
- Database :
- OpenAIRE
- Journal :
- Current Genetics
- Accession number :
- edsair.doi.dedup.....0968bcf55536a5bec3c23c7fae4e2e00
- Full Text :
- https://doi.org/10.1007/s00294-009-0252-z