Melanin synthesis using a new melanogenic strain of Flavobacterium kingsejongi and a recombinant strain of Escherichia coli expressing 4-hydroxyphenylpyruvate dioxygenase from F.kingsejongi
- 주제(키워드) 멜라닌 , Melanins
- 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/000000031323
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Melanins are a heterologous group of biopolymeric pigments synthesized by diverse prokaryotes and eukaryotes and are widely utilized as bioactive materials and functional polymers in the biotechnology industry. Here, we report high-level melanin production using a new melanogenic Flavobacterium kingsejongi strain and a recombinant Escherichia coli overexpressing F. kingsejongi 4-hydroxyphenylpyruvate dioxygenase (HPPD). Melanin synthesis by the F. kingsejongi strain was confirmed via melanin synthesis inhibition tests, melanin solubility tests, genome analysis, and structural analysis of the purified melanin from both wild-type F. kingsejongi and recombinant E. coli expressing F. kingsejongi HPPD. The activity of F. kingsejongi HPPD was demonstrated via in vitro assays with 6×His-tagged and native forms of HPPD. Bioreactor fermentation of F. kingsejongi produced a large amount of melanin with a titer of 6.07 ± 0.32 g/L, a conversion yield of 60% (0.6 ± 0.03 g melanin per gram tyrosine), and a productivity of 0.03 g/L·h, indicating its potential for industrial melanin production. Additionally, bioreactor fermentation of recombinant E. coli expressing F. kingsejongi HPPD produced melanin at a titer of 3.76 ± 0.30 g/L, a conversion yield of 38% (0.38 ± 0.03 g melanin per gram tyrosine), and a productivity of 0.04 g/L·h. Both strains showed sufficiently high fermentation capability to indicate their potential as platform strains for large scale bacterial melanin production.
more목차
1. Introduction 1
2. Materials and Methods 5
2.1 Strains, media, and culture conditions 5
2.2 Melanin synthesis inhibition test 5
2.3 Extraction and analysis of melanin 6
2.4 Characterization of melanin extracted from culture broth 6
2.5 Genome mining and heterologous expression of proteins involved in melanin biosynthesis 7
2.6 Expression and purification of the F. kingsejongi HPPD-encoding gene using E coli 8
2.7 In vitro HPPD activity assay 9
2.8 Analysis of mRNA expression level of HPPD 10
2.9 Phylogenetic analysis of HPPD 11
2.10 Batch fermentation of F. kingsejongi in a 5-L bioreactor 11
2.11 Batch fermentation of recombinant E coli expressing HPPD in a 5-L bioreactor 12
2.12 Analytical methods 12
3. Results and Discussion 16
3.1 Characteristics of the brownish-black pigment from F. kingsejongi 16
3.2 Chemical characterization of the brownish-black pigment from F. kingsejongi 19
3.3 Identification and heterologous expression of the putative eumelanin-synthesizing gene of F. kingsejongi 21
3.4 In vitro activity of purified 6×His-tagged HPPD and native HPPD in crude protein extract 26
3.5 Bioreactor batch fermentation of F. kingsejongi 30
3.6 Bioreactor batch fermentation of recombinant E coli expressing HPPD 33
4. Conclusion 36
5. References 37
ABSTRACT IN KOREAN 43
