The ultimate goal in the field of metabolic engineering is improving cellular processes in a rational manner using engineering design principles and molecular biology techniques. The syntheses of several industrially useful compounds are cofactor-dependent. The reducing equivalent NADPH is required in several enzymatic reactions leading up to the synthesis of high-value compounds like polymers, chiral alcohols, and antibiotics. However, it’s a highly costly compound with limited intracellular availability. This study focuses on the genetic manipulation of a whole-cell system using the two transhydrogenase isoforms pntAB and udhA. Two model systems are used: 1) the production of (S)-2-chloropropionate and 2) the production of poly(3-hydroxybutyrate). Results suggest that the presence of udhA increases product yield and NADPH availability while the presence of pntAB has the opposite effect. A maximum product yield of 1.4 mole-product/mole-glucose was achieved aerobically in a pntAB-deletion strain with udhA overexpression, a 150% improvement over the wild-type control strain.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Alguns organismos podem biossintetizar L-prolina (L-Pro) principalmente via L-glutamato (L-Glu). A Δ1-pirrolina-5-carboxilato (P5C) sintase (P5CS) converte o L-Glu ao intermediário da via, o P5C que uma vez formado é reduzido a L-Pro via P5C redutase (P5CR). Nesse trabalho, verificou-se a possível ocorrência dessa via em Tritryps. Em Trypanosoma cruzi toda a via é operativa, em Trypanosoma brucei está ausente, enquanto que em Leishmania amazonensis acontece parcialmente. A biossíntese de L-Pro em T. cruzi é abordada com mais detalhes. Ambas as enzimas da via foram expressas e tiveram sua localização determinada no citosol, compartimento distinto das enzimas do catabolismo de L-Pro. Ainda realizou-se a caracterização bioquímica da TcP5CR, que curiosamente sofre inibição incompetitiva pelo NADPH, e por isso é candidata a regular esse metabolismo. Por fim, evidenciou-se a saída do P5C da mitocôndria, possibilitando a existência de um mecanismo de transidrogenação via a interconversão de L-Pro em P5C e vice versa. / Some organisms biosynthesize L-proline (L-Pro) mainly from L-glutamate (L-Glu). The Δ1-pyrroline-5-carboxylate (P5C) synthase (P5CS) converts L-Glu into the intermediate P5C, which in turn, is reduced to L-Pro by a P5C reductase (P5CR). In this study, we verified the possible occurrence of this pathway in Tritryps. In Trypanosoma cruzi the entire route is operative. In Trypanosoma brucei, is absent, while Leishmania amazonensis this pathway is partially operative. In T. cruzi, both enzymes were expressed and had their location determined in the cytosol, differently from those of the catabolism of L-Pro. In addition, the biochemical characterization of TcP5CR showed an uncompetitive inhibition by NADPH, and therefore it is a candidate to be a regulation point of the pathway. Finally, it was observed the exit of PC5 from the mitochondria, a condition for the existence of a transhydrogenation mechanism via the interconversion between Pro and P5C.

Orientador: Helena Coutinho Franco de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T17:01:10Z (GMT). No. of bitstreams: 1 Rovani_JulianaCristine_M.pdf: 3138644 bytes, checksum: 95185175ce4721f82feadc47122e6ed0 (MD5) Previous issue date: 2014 / Resumo: A transidrogenase de nucleotídeo de nicotinamida mitocondrial (NNT) é a principal fonte geradora de NADPH da organela, o qual é utilizado pelo sistema antioxidante das mitocôndrias. Estudos prévios mostraram que a mutação nessa enzima está associada a alterações do estado redox de mitocôndrias e alterações do metabolismo glicídico e lipídico em animais C57BL6/J provenientes do Jackson Laboratory (B6-J). A síndrome metabólica é caracterizada pela presença de no mínimo três distúrbios cardio-metabólicos associados dentre obesidade visceral, dislipidemia, intolerância à glicose e/ou resistência à insulina e hipertensão. A restrição calórica tem sido indicada para prevenir e tratar estes distúrbios metabólicos. Compostos sintéticos têm sido testados como potenciais miméticos da restrição calórica. O 2,4-dinitrofenol (DNP) é uma droga que aumenta o gasto energético por provocar redução da eficiência da fosforilação oxidativa e aumentar o metabolismo oxidativo. Neste trabalho, demonstramos que camundongos com mutação no gene da NNT (B6-J) apresentaram aumento de adiposidade visceral, aumento do teor de triglicérides hepático, intolerância à glicose, resistência à insulina e hipersecreção de insulina pelas ilhotas pancreáticas isoladas e in vivo, quando comparados aos camundongos que tem a NNT intacta (B6-UNI). Diante destes distúrbios metabólicos, submetemos os animais à restrição alimentar (RA) ou ao tratamento com DNP durante 3 meses. Observamos que os animais B6-J sob RA apresentaram redução da adiposidade, esteatose hepática corrigida, melhora da resistência à insulina e diminuição da secreção de insulina. Por outro lado, o tratamento com DNP nos animais B6-J não modificou o metabolismo lipídico, reduziu a insulinemia de jejum e a secreção de insulina e melhorou a sensibilidade periférica à insulina, mas não alterou a tolerância à glicose. Concluímos que a linhagem portadora da deleção do gene da NNT tem maior predisposição à síndrome metabólica, e que, ambos os tratamentos de RA e DNP corrigiram, pelo menos parcialmente, os distúrbios metabólicos dos animais B6-J, sendo que a RA foi mais efetiva que o DNP / Abstract: The nicotinamide nucleotide transhydrogenase (NNT) catalyses the production of NADPH, that is consumed by the mitochondrial antioxidant enzymatic system. Previous studies have shown that NNT mutation causes redox abnormalities in mitochondria and alterations in glucose and lipid metabolism in C57BL6/J mice from The Jackson Laboratory. Metabolic syndrome is characterized by an association of cardio-metabolic disturbances such as visceral obesity, dyslipidemias, glucose intolerance and/or insulin resistance and hypertension. Caloric restriction has been recommended to prevent or treat such metabolic disturbances. Synthetic compounds have been tested as putative mimetic for caloric restriction. The 2,4 dinitrophenol (DNP) is one of this drugs that increase the metabolic rates due to a reduction in the oxidative phosphorylation efficiency and elevation of oxidative metabolism. In this study we demonstrated that mice that carry a NNT gene mutation (B6-J) exhibit increased visceral adiposity, increased liver triglyceride content, glucose intolerance, insulin resistance, and insulin hypersecretion, when compared to control mice with functional NNT. In face of these disturbances, mutant B6-J mice were submitted to a food restriction (FR) or treatment with DNP during 3 months. We observed that B6-J mutant mice under FR present a reduction in adiposity and liver steatosis, improvement of glucose tolerance and insulin resistance and normalization of insulin secretion. After DNP treatment, B6-J mice showed no alterations in lipid disturbances but improved insulin resistance and insulin secretion. These findings suggest that 1- the mouse substrain that carries NNT mutation is predisposed to develop metabolic syndrome, 2- both treatments, FR and DNP, correct, at least partially, the metabolic disturbances of B6-J mutant mice, and 3- FR treatment was more effective than DNP / Mestrado / Fisiologia / Mestra em Biologia Funcional e Molecular

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 197 pages. Includes vita. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references (leaves 188-197). Also available online.

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