Construction of a platform mutant strain of Wickerhamomyces ciferrii for the production of rare sphingoid base
- 주제(키워드) rare sphingoid base , wickerhamomyces ciferrii
- 주제(DDC) 547
- 발행기관 아주대학교 일반대학원
- 지도교수 이평천
- 발행년도 2025
- 학위수여년월 2025. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000034369
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Ceramides, composed of sphingoid bases and fatty acids, are widely used in the food, pharmaceutical, and cosmetic industries. Because of their diversity, most ceramides are chemically synthesized or extracted from plants. To date, many efforts have been made to improve the production of tetraacetyl phytosphingosine (TAPS) in Wickerhamomyces ciferrii, as it is a natural producer of this sphingoid base. However, the production of other sphingoid bases, such as sphinganine (Sa) and sphingosine (So), has been rarely studied in microbes including W. ciferrii. To produce rare sphingoid bases in W. ciferrii, we developed a screening system with fluorescein sodium to isolate strains capable of producing rare sphingoid bases from randomly mutagenized library. Using this screening system, we successfully isolated sphingosine-producing mutant P41C3 strain. Furthermore, batch fermentation with the sphingosine- producing mutant P41C3 strain was conducted. The results showed that the production of tetraacetyl phytosphingosine, which is competitor of sphinganine and sphingosine in metabolic pathway, decreased by 50.4% (mid- log phase) and 69.4% (stationary phase) compared to the production of the wild-type strain. The production of rare sphingoid bases reached 1.5 mg-So/g- DCW and 0.4 mg-Sa/g-DCW in the mid-log phase, and 2.73 mg-So/g-DCW and 4.38 mg-Sa/g-DCW in the stationary phase. In conclusion, we successfully developed the first screening system with fluorescein sodium to isolate mutant strains producing rare sphingoid bases, such as sphinganine and sphingosine. Using this screening system, the sphingosine-producing mutant P41C3 strain was isolated, and its production was confirmed through batch fermentation.
more목차
1. Introduction 1
2. Materials and Methods 6
2.1 Strains, mutant library generation, and culture conditions 6
2.2 Selective reactivity assay between sphingoid bases and fluorescein sodium 7
2.3 Fluorescein sodium toxicity assay on cells 7
2.4 Fluorescence-Activated Cell Sorting (FACS) Analysis 8
2.5 Confocal microscopy analysis 8
2.6 Sequence variation analysis in sphingoid base pathway genes 9
2.7 Fatty acid composition analysis by GC-MS 10
2.8 Transcriptional analysis 10
2.9 Batch fermentation 11
2.10 Extraction of sphingoid bases 11
2.11 HPLC analysis 12
3. Results 15
3.1 Selective reactivity between fluorescein sodium and sphingoid bases, and its effect on cell viability 15
3.2 Optimization of staining conditions for FACS analysis 19
3.3 Modification of method for rapid extraction of sphingoid bases 21
3.4 Fluorescein sodium-based screening to isolate rare sphingoid bases-producing mutant strains 24
3.5 Characterization of the sphingosine-producing mutant strain 28
3.6 Batch fermentation of the sphingosine-producing mutant strain 33
4. Discussion 36
5. References 38
ABSTRACT IN KOREAN 43
