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대장균에서 Pantoea agglomerans와 Corynebacterium glutamicum 유래 카로틴노이드 생합성 대사회로의 재구축
- Author(s)
- Song, Gyu Hyeon
- Advisor
- 이평천
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2011-08
- Language
- eng
- Alternative Abstract
- Enzymes efficiently catalyze the conversion of a vast number of molecular structures. Such catalysts of biological systems are models of energy efficient, environmentally friendly chemical agents, because all do their work under mild conditions (in water, at room temperature and atmospheric pressure) and generate few waste products (1). Many efforts to use biological system for production of valuable compounds have been established, especially in field of metabolic engineering. Metabolic engineering is the science that combines systematic analysis of metabolic and other pathways with molecular biological techniques to improve cellular properties by designing and implementing rational genetic modifications (18). To explicate the whole biological pathway, it also includes many tools, such as molecular cloning, synthetic biology, protein engineering, proteomics, and metabolomics. Most of these studies focused on making an improved product yield. For example, overexpression of rate-limiting enzyme in biosynthesis pathway resulted in improved accumulation of derivative molecules. For example, overexpression of idi (isopentenyl diphosphate isomerase) is the key engineering for precursor availability and improved the yield of carotenoids production in metabolically engineered E. coli (21, 23). Otherwise, reordering biosynthetic genes in the natural biosynthetic cluster to optimize the production of end-product (30) or overexpression of specific enzymes to optimize the enzymic levels for minimize the accumulation of intermediates and increase the yield of the desired end-product (36). Carotenoids are derivatives from isoprenoid that plays various role in nature with tremendous diversity (32). Previously, carotenoids were interested as natural pigment. They exhibit a characteristic type of absorbance spectrum with three peaks in the major band phytoene, the first C40 carotenoid precursor to later carotenoids, absorbs in the UV range (3). For examples, carotenoids occur in the tomato fruit, whose characteristic red color is due to C40 acyclic carotenoid lycopene and in the orange, whose yellow color is due to C40 cyclic carotenoid zeaxanthin. But more recent researches show that it has antioxidizing and anticarcinogenic activity as cosmeceutical, pharmaceutical compounds (9, 29, 39). They are synthesized by photosynthetic and some non-photosynthetic organisms (4). More than 700 carotenoids were isolated and identified (14, 47). Naturally occurring carotenoids biosynthetic pathway are consists of C30, C40, and C50 carotenoid biosynthetic pathway (Figure 1) (23) based on carbon number of their basic structure. C30 carotenoids were synthesized by condensation of two molecules of farnesyl diphosphate (C15, FPP). C30 carotenoids are much less widespread, having been found only in a small number of bacteria such as Staphylococcus, Streptococcus, Heliobacterium, and Methylobacterium species (45). While on the other, C40 carotenoids were synthesized by condensation of two molecules of geranylgeranyl diphosphate (C20, GGPP) and are common in nature. C50 carotenoids are elongated form of C40 carotenoids, synthesized by prenylation of lycopene. Here, C40 and C50 carotenogenic pathway were reconstructed from Pantoea agglomerans and Corynebacterium glutamicum into heterologous host, E. coli. The genes encoding CrtE (GGPP synthase), CrtB (Phytoene synthease), CrtI (phytoene desaturase), CrtY (lycopene cyclase), CrtZ (β-carotene hydroxylase) and CrtX (zeaxanthin glucosyltransferase) were isolated from P. agglomerans carotenogenic gene cluster which produce zeaxanthin diglucoside in original host strain, and genes encoding CrtE, CrtB1, CrtB2, CrtI1, CrtI2, CrtEb (lycopene elongase) and CrtYeYf (C50 carotenoid epsilon cyclase) were isolated from C. glutamicum carotenogenic gene cluster which produce decaprenoxanthin and derivates in original host strain. These genes were reconstructed in E. coli, after that, their carotenoids profiles and functionality of enzyme was analyzed in vivo or in vitro.
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- Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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