Boergel, Daniel and van den Berg, Marco and Hueller, Thomas and Andrea, Heiko and Liebisch, Gerhard and Boles, Eckhard and Schorsch, Christoph and van der Pol, Ruud and Arink, Anne and Boogers, Ilco and van der Hoeven, Rob and Korevaar, Kees and Farwick, Mike and Koehler, Tim and Schaffer, Steffen (2012) Metabolic engineering of the non-conventional yeast Pichia ciferrii for production of rare sphingoid bases. METABOLIC ENGINEERING, 14 (4). pp. 412-426. ISSN 1096-7176,
Full text not available from this repository. (Request a copy)Abstract
The study describes the identification of sphingolipid biosynthesis genes in the non-conventional yeast Pichia ciferrii, the development of tools for its genetic modification as well as their application for metabolic engineering of P. ciferrii with the goal to generate strains capable of producing the rare sphingoid bases sphinganine and sphingosine. Several canonical genes encoding ceramide synthase (encoded by PcLAG1 and PcLAF1), alkaline ceramidase (PcYXC1) and sphingolipid C-4-hydroxylase (PcSYR2), as well as structural genes for dihydroceramide Delta(4)-desaturase (PcDES1) and sphingolipid Delta(8)-desaturase (PcSLD1) were identified, indicating that P. ciferrii would be capable of synthesizing desaturated sphingoid bases, a property not ubiquitously found in yeasts. In order to convert the phytosphingosine-producing P. ciferrii wildtype into a strain capable of producing predominantly sphinganine, Syringomycin E-resistant mutants were isolated. A stable mutant almost exclusively producing high levels of acetylated sphinganine was obtained and used as the base strain for further metabolic engineering. A metabolic pathway required for the three-step conversion of sphinganine to sphingosine was implemented in the sphinganine producing P. ciferrii strain and subsequently enhanced by screening for the appropriate heterologous enzymes, improvement of gene expression and codon optimization. These combined efforts led to a strain capable of producing 240 mg L-1 triacetyl sphingosine in shake flask, with tri- and diacetyl sphinganine being the main by-products. Lab-scale fermentation of this strain resulted in production of up to 890 mg kg(-1) triacetyl sphingosine. A third by-product was unequivocally identified as triacetyl sphingadienine. It could be shown that inactivation of the SLD1 gene in P. ciferrii efficiently suppresses triacetyl sphingadienine formation. Further improvement of the described P. ciferrii strains will enable a biotechnological route to produce sphinganine and sphingosine for cosmetic and pharmaceutical applications. (C) 2012 Elsevier Inc. All rights reserved.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | TANDEM MASS-SPECTROMETRY; CODON ADAPTATION INDEX; SACCHAROMYCES-CEREVISIAE; HANSENULA-CIFERRI; ALKALINE CERAMIDASE; CANDIDA-ALBICANS; STRATUM-CORNEUM; GENE; CLONING; SPHINGOLIPIDS; Pichia ciferrii; Sphingolipids; Sphinganine; Sphingosine; Metabolic engineering; Codon optimization |
| Subjects: | 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Medicine > Lehrstuhl für Klinische Chemie und Laboratoriumsmedizin |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 12 May 2020 09:56 |
| Last Modified: | 12 May 2020 09:56 |
| URI: | https://pred.uni-regensburg.de/id/eprint/18546 |
Actions (login required)
 |
View Item |
