Construction of crocin biosynthesis pathway in Saccharomyces cerevisiae
- 주제(키워드) crocin
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 이평천
- 발행년도 2022
- 학위수여년월 2022. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031684
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Crocins belong to the apocarotenoid glycosides and are the main bioactive components in saffron, derived from the stigmas of Crocus sativus. Under the need for efficient and economical methods for producing crocins, microbial production enhances the possibility of fabrication of crocins. Four major heterologous enzymes were introduced in S.cerevisiae for simulating the crocin biosynthetic pathway. β-carotene hydroxylase (CrtZ), carotenoid cleavage dioxygenase (CCD), aldehyde dehydrogenase (ALDH), and two types of UDP-glucosyltransferases (UGTs) were expressed, including novel enzymes that were isolated by our group. A qualitative-quantitative analysis of the precursor was performed at each step to find the optimal catalytic activities among candidate enzymes from different organism sources. As a result, the metabolic engineered S.cerevisiae strain produced 65.23 ± 2.97μg/L of crocin-4 in flask cultivation. To increase the supply of UDP-glucose, two genes named pgm2, ugp1 involved in endogenous uridine diphosphate glucose pathway were over-expressed respectively in S. cerevisiae. As a result, overexpression of a pgm2 gene encoding phosphoglucomutase enhanced 2.1 fold increase in crocin-4 from 65.23 ± 2.97μg/L to 136.17 ± 5.89μg/L in flask cultivation. Finally, a batch culture operated under the conditions (20℃, pH 5.5, and a DO of 40%) enhanced up to 204.25 ± 8.83 μg/L of crocin-4 in a bioreactor. To the best of our knowledge, our study presents the first crocin-producing S. cerevisiae cell factory.
more목차
1. Introduction 1
2. Materials and Methods 4
2.1 Strains and culture conditions 4
2.2 Plasmid construction for the expression of crocin pathway enzymes 5
2.3 Construction of β-carotene producing strain 7
2.4 Integration CrtZ and CCD genes into the chromosome 8
2.5 Transcriptional analysis 9
2.6 Batch fermentation 9
2.7 Extraction and analysis of carotenoids 10
3. Results 17
3.1 Construction of zeaxanthin biosynthetic pathway in S.cerevisiae 17
3.2 Confirmation of CsCCD2 bioactivity in S. cerevisiae 19
3.3 Comparison of ALDHs for crocetin production 21
3.4 Finding optimal crocetin-glucosyltransferase for efficient conversion to crocins 23
3.5 In vivo production of crocin-4 in S. cerevisiae 25
3.6 Enhanced production of crocin-4 by increasing the supply of UDP-glucose 27
3.7 Batch fermentation of crocin-producing strains 30
4. Discussion 32
5. References 33
