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Investigation of the effects of culture conditions on cell growth and tetraacetylphytosphingosine production in Wickerhamomyces ciferrii mutant strain

  • 은성욱 (아주대학교 일반대학원 )

초록/요약

Wickerhamomyces ciferrii is a non-conventional yeast strain characterized by the production and secretion of large amounts of tetraacetylphytosphingosine (TAPS). TAPS is an industrially valuable substance that can be easily converted into ceramide through deacetylation and can be used in the pharmaceutical and cosmetics fields. W. ciferrii is typically cultured in complex medium and there are no reports in the defined medium for TAPS production. The complex medium provides sufficient nutrients but has the disadvantage of not being able to identify the medium components. To investigate which components, affect TAPS production and to improve additional TAPS productivity, the medium and culture conditions were optimized for the W. ciferrii mutant 736 and recombinant 736. To develop a defined medium for mutant 736, single omission cultivation and bioreactor fermentation were performed based on reference defined medium. Among the defined medium components (trace metal, vitamins), TAPS production was increased in the medium without ZnSO4. In the new medium, TAPS production (1.85 ± 0.1 g/L conversion yield of 0.046 g-TAPS/g-glycerol) was increased by approximately 28 % compared to that in the complex medium (1.45 ± 0.02 g/L, conversion yield of 0.036 g-TAPS/g-glycerol) at the bioreactor level. Additionally, a recombinant 736, expressing TAPS biosynthetic pathway genes, was cultured in constructed medium. Compared to the mutant 736 (1.05 ± 0.1 g/L), TAPS has increased by up to 50 % in recombinant 736 (1.53 ± 0.1 g/L). Since culture optimization for TAPS production in the defined medium is in the early stages of development, mathematical and statistical medium optimization can increase TAPS productivity. Further optimization of the expression level of TAPS biosynthetic pathway genes should improve the current titer of TAPS.

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초록/요약

위커하모마이세스 시페리는 다량의 테트라아세틸피토스핑고신(TAPS)을 생산 및 분비하는 것을 특징으로 하는 비전통 효모 균주이다. TAPS 는 탈아세틸화를 통해 세라마이드로 쉽게 전환될 수 있는 산업적으로 가치가 있는 물질로 의약품과 화장품 분야에서 활용이 가능하다. 위커하모마이세스 시페리는 일반적으로 complex 배지에서 배양되며 TAPS 생성에 대한 defined배지에 대한 보고는 없다. Complex 배지는 충분한 영양분을 제공하지만, 배지 성분을 정확히 알 수 없다는 단점이 있다. TAPS 생산에 영향을 미치는 성분이 무엇인지 조사하고 추가적인 TAPS 생산성을 향상시키기 위해 배지 및 배양 조건을 W. ciferrii 돌연변이 736 과 재조합 736 에 최적화하였다. W. ciferrii 돌연변이 736 에 대한 defined 배지를 개발하기 위해 기존 알려진 defined 배지를 기반으로 단일 생략 배양과 생물반응기 발효를 수행하였다. 정의된 배지 성분(트레이스메탈, 비타민) 중 ZnSO4 가 없는 배지에서 TAPS 생산이 증가하였다. 새로운 배지에서 TAPS 생산은 생물반응기 수준에서 complex 배지 (1.45 ± 0.02 g/L)에 비해 약 28% (1.85 ± 0.1 g/L) 증가하였다. 또한, TAPS 생합성 경로 유전자를 발현하는 재조합 736 을 새로운 배지에서 배양한 결과, TAPS 생산성은 기존 돌연변이 736 (1.05 ± 0.1 g/L)과 비교해 재조합 736 (1.53 ± 0.1 g/L)에서 최대 50% 증가했다. defined 배지에서의 TAPS 생산에 대한 배양 최적화는 개발 초기 단계이기 때문에 수학적, 통계적 배지 최적화는 TAPS 생산성을 증가시킨다. TAPS 생합성 경로 유전자의 발현 수준의 추가 최적화는 W. ciferrii 에서 TAPS 의 생산량을 증가시킬 것으로 생각된다.

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목차

1. Introduction 1
2. Materials and Methods 5
2.1 Strains, and culture conditions 5
2.2 Extraction and analysis of TAPS 5
2.3 96 well plate culture for C/N ratio 6
2.4 Omission test: trace metals and vitamins 6
2.5 Flask fermentation procedures 7
2.6 Plasmid construction for the expression of TAPS pathway enzymes 7
2.7 Transformation of W. ciferrii mutant 736 and flask cultivation 8
2.8 Transcriptional analysis 8
2.9 Batch fermentation under different conditions 9
2.10 Comparative fermentation with complex and modified defined medium 9
2.11 Analytical methods 9
3. Results and Discussion 13
3.1 Optimization of the C/N ratio 13
3.2 Single omission cultivation: vitamins and trace metals 15
3.3 TAPS production by the wild-type and mutant 736 in different concentrations of ZnSO4 17
3.4 Effect of different culture conditions on TAPS production 20
3.5 Comparative fermentation with complex and modified defined medium 22
3.6 Enhanced TAPS production of recombinant mutant 736 24
4. Conclusion 26
5. References 27

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