Journal
CURRENT GENETICS
Volume 55, Issue 4, Pages 381-389Publisher
SPRINGER
DOI: 10.1007/s00294-009-0252-z
Keywords
Pichia ciferrii; LIG4; Non-homologous end joining; Gene targeting
Categories
Funding
- German Federal Ministry of Education and Research
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available