Development of Corynebacterium glutamicum as a platform strain for the biosynthesis of crocetin
- 주제(키워드) Corynebacterium glutamicum , crocetin
- 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/000000034468
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Crocetin derived from Gardenia jasminoides and Crocus sativus has been reported to have significant potential in the food and pharmaceutical markets. Due to the limitations of time and cost in the plant extraction process, microbial production is proposed as an alternative. De novo biosynthesis of crocetin has been reported in Escherichia coli and Saccharomyces cerevisiae. However, additional strains have yet to be identified as hosts for crocetin production. Corynebacterium glutamicum is a natural carotenoid producer and is widely used industrially for amino acid production, making it a strong candidate as a novel host strain for crocetin biosynthesis. In this study, We aimed to develop a genetically engineered C. glutamicum strain for crocetin production. Initially, we constructed a lycopene- overproducing strain by deleting a competing pathway and overexpressing 1- deoxy-D-xylulose-5-phosphate synthase and geranylgeranyl diphosphate synthase. This strain has a 71.3-fold higher lycopene production than the wild type. Subsequently, lycopene β-cyclase from Pantoea agglomerans (CrtYPa) was introduced into the lycopene-producing strain. Furthermore, beta- carotene hydroxylase from P. agglomerans (CrtZPa) was introduced into the β-carotene-producing strain. The conversion of β-carotene to zeaxanthin was identified as a bottleneck due to the extremely low zeaxanthin titer. Thus, we overexpressed CrtZPa via an episomal plasmid, producing 33.80 ± 0.28 mg/L zeaxanthin, which represents the highest reported zeaxanthin production in C. glutamicum. Finally, carotenoid cleavage dioxygenase 2 from Crocus sativus and aldehyde dehydrogenase from Synechococcus elongatus were introduced into the zeaxanthin-producing strain. Despite confirmation of their expression by RT-PCR, neither crocetin dialdehyde nor crocetin has yet been detected.
more목차
1. Introduction 1
2. Materials and Methods 4
2.1 Strains and culture conditions 4
2.2 Construction of plasmids for the expression and integration 4
2.3 Transformation 5
2.4 Genomic engineering 5
2.5 Transcriptional analysis 6
2.6 Extraction of carotenoids 6
2.7 TLC analysis 6
2.8 HPLC-DAD analysis 7
3.Results 13
3.1 Construction of enhanced lycopene-producing C. glutamicum strains 13
3.2 In vivo assessment of lycopene β-cyclase activity from different organisms via β-carotene production in C. glutamicum 18
3.3 Chromosomal integration of crtYPa gene into the L3 strain 20
3.4 In vivo assessment of beta-carotene hydroxylase activity from different organisms via zeaxanthin production in C. glutamicum 22
3.5 Chromosomal integration of crtZPa gene into the B1 strain 24
3.6 Enhancement of zeaxanthin production via crtZPa expression from a plasmid in C. glutamicum 26
3.7 Chromosomal integration of CCD2 and aldH gene into the Z1 strain 28
4. Discussion 30
5. References 32
ABSTRACT IN KOREAN 37
