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Title:In vitro production, purification and metabolism of tomato carotenoids
Author(s):Lu, Chi-Hua
Director of Research:Erdman, John W.
Doctoral Committee Chair(s):de Mejia, Elvira G.
Doctoral Committee Member(s):Erdman, John W.; Cadwallader, Keith R.; Jin, Yong-Su
Department / Program:Food Science & Human Nutrition
Discipline:Food Science & Human Nutrition
Degree Granting Institution:University of Illinois at Urbana-Champaign
Response Surface Methodology (RSM)
Carbon-13 (13C)
Escherichia coli (E. coli)
Carotene-9’,10’-monooxygenase (CMO-II)
Abstract:Tomato consumption has been correlated with decreased risks of chronic diseases like prostate cancer and cardiovascular disease. Among many nutrients and phytochemicals in fresh tomatoes and tomato products, carotenoids are thought to be the bioactive components responsible for disease prevention. Many epidemiological studies, clinical trials, animal studies, and in vitro studies have focused on the most abundant tomato carotenoid, lycopene. However, the absorption, distribution and metabolism of phytoene and phytofluene, the precursors of lycopene and the 2nd- and the 3rd-most abundant tomato carotenoids, have not been fully studied. With the discovery of the human carotenoid cleavage enzymes carotene-15’,15’-monooxygenase (CMO-I) and carotene-9’,10’-monooxygenase (CMO-II), enzymatic degradation is proposed to be a mechanism of tomato carotenoid metabolism. In order to elucidate the metabolism and excretion routes of absorbed tomato carotenoids, a tracer system must be established to assist in locating the parent molecules, identifying metabolic products and potentially calculating kinetics/dynamics models. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. The first section of this research was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory HPLC separation. We employed response surface methodology (RSM), which combines fractional factorial design and a second-degree polynomial model. Tomato cells were homogenized with ethanol, saponified by KOH, extracted with hexane, and the lycopene content was analyzed by HPLC-PDA. We varied five factors at five levels: ethanol volume (1.33-4 mL/g); homogenization period (0-40 s/g); saturated KOH solution volume (0-0.67 mL/g); hexane volume (1.67-3 mL/g); and vortex period (5-25 s/g). Ridge analysis by SAS suggested that the optimal extraction procedure to extract 1 g of tomato cells was at 1.56 mL ethanol, 28 s homogenization, 0.29 mL KOH, 2.49 mL hexane and 17.5 s vortex. These optimal conditions predicted by RSM were confirmed to enhance lycopene yield from standardized tomato cell cultures by more than three fold. Furthermore, a bioengineered Escherichia coli model for the laboratory-scale production of stable isotope-labeled carotenoids was developed. Carotenoid biosynthetic genes from Enterobacter agglomerans were introduced into the BL21Star(DE3) strain to yield lycopene. Over 96% of accumulated lycopene was in the all-trans isoform, and the molecules were highly enriched with 13C by 13C-glucose dosing. In addition, error-prone PCR was used to create a phytoene-accumulating strain, which maintained the transcription of phytoene synthase (crtB). Phytoene molecules were also highly enriched with 13C when 13C-glucose was the only carbon source. The development of this production model will provide carotenoid researchers a source of labeled materials and to further investigate the metabolism and biological functions of these carotenoids. To date, there is no experimental evidence to demonstrate the biological function of human CMO-II; therefore, Escherichia coli was selected to express this enzyme. The full-length human CMO-II sequence and a truncated CMO-II sequence were expressed, but the expressed proteins were insoluble. Tagging with NusA did not improve its solubility. E. coli strain Origami 2 and co-expression of CMO-II with chaperonins were also used to provide a protein-folding friendly environment, but the majority of CMO-II remained insoluble. Furthermore, denaturation, purification and refolding procedures were used to rescue CMO-II, but the refolded enzymes did not degrade the carotenoid substrate lutein. In conclusion, other expression hosts rather than E. coli might be better platforms to express soluble human CMO-II and further elucidate its role in carotenoid metabolism. Overall, this research explored suitable production systems to produce isotopically labeled carotenoids and piloted the expression of the human enzyme CMO-II in E. coli. With the tools developed in this research, soon we will be able to discover the details of the metabolism of tomato carotenoids and further improve the human health.
Issue Date:2011-08-26
Rights Information:Copyright 2011 Chi-Hua Lu
Date Available in IDEALS:2011-08-26
Date Deposited:2011-08

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