In eukaryotes, L-carnitine is involved in energy metabolism, where it facilitates b-oxidation of fatty acids. Carnitine acyltransferases catalyze the reversible conversion of acyl-CoA and carnitine to acylcarnitine and free CoA. There are three carnitine acyltransferase families, which differ in their acyl-chain length selectivity: carnitine palmitoyltransferases (CPTs) catalyze long-chain fatty acids, carnitine octanoyltransferase (COT) prefers medium-chain fatty acids, and carnitine acetyltransferase (CrAT) uses short-chain acyl-CoAs. In this study, we attempted to identify the amino acid residues responsible for acyl-CoA specificity in the acyltransferase family through structure-based mutagenesis studies. We identified an amino acid (Met564 in rat CrAT) that is critical to fatty acyl chain-length specificity. A CrAT protein carrying the M564G mutation behaved as if its natural substrates were medium-chain acyl-CoAs, similar to COT. In the reverse case, mutation of the orthologous glycine (Gly553) to methionine in COT decreased activity towards its natural substrates, medium-chain acyl-CoAs, and increased activity towards short-chain acyl-CoAs. A second putative amino acid involved in acyl-CoA specificity was identified (Asp356 in rat CrAT), and the double CrAT mutant D356A/M564G behaved as a pseudo-CPT in terms of substrate specificity. Three-dimensional models revealed a deeper hydrophobic cavity for the binding of acyl groups in both CrAT mutants in the same position as the shallow cavity in the wt enzyme. Furthermore, we studied the effect of C75-CoA, a potent and competitive inhibitor of CPT I, on CrAT activity. C75-CoA occupies the same pocket in CPT I as palmitoyl-CoA, suggesting an inhibitory mechanism based on mutual exclusion. To determine whether this inhibitor would fit in the open hydrophobic pocket formed in CrAT mutants M564G and D356A/M564G, we carried out competitive inhibition assays. Our experiments showed that while C75-CoA is a potent inhibitor of CrAT mutants M564G and D356A/M564G, it has no effect on wt CrAT. Choline acetyltransferase (ChAT) catalyzes a similar reaction to CrAT, with the difference that in ChAT the acetyl group from acetyl-CoA is transferred to choline instead of carnitine. Cronin (1998) successfully redesigned ChAT to use carnitine instead of its natural substrate choline. In the present study, our aim was to achieve the opposite, that is, to redesign rat CrAT specificity from carnitine to choline. We prepared a mutant CrAT that incorporates four amino acid substitutions, and the resulting mutant shifted the catalytic discrimination between L-carnitine and choline in favour of the latter substrate. The food industry is interested in the production of esters for use as flavouring compounds; for example, esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced in an enzyme-catalyzed reaction between a higher alcohol and an acyl-CoA molecule. Since CrAT is responsible for the modulation of the acyl-CoA/CoA ratio, we hypothesized that overexpression of this enzyme could modify ester production in yeast. Therefore, we overexpressed CrAT in yeast and analysed its effect on ester production during alcoholic fermentation. Compared with control cells, overexpression of CrAT caused a significant reduction in the production of some esters, including the important flavour components ethyl acetate and 3-methyl-butyl acetate.In conclusion, the amino acid substitutions in rat CrAT and COT in this study reveal several residues that are involved in substrate recognition and provide insight into the molecular requirements for their correct positioning in order to achieve efficient catalysis. These results not only help us to understand the structure-function relationship within the acyltransferase family, but may also facilitate studies on obesity, non-insulin dependent diabetes, and patients with defective â-oxidation. Moreover, our results open the possibility of biotechnological applications of the enzymes of the carnitine acyltransferase family in the wine industry.

L’arròs es un cultiu bàsic, consumit per mes de 3 billons de persones. Es una font pobra en ferro (Fe) i zinc (Zn) per la qual cosa les poblacions pobres que depenen de l’arros per a la seva supervivència, sofreixen d’anèmia por deficiència de ferro (IDA) i zinc (ZnD). La desnutrició es un repte important, especialment en els països en desenvolupament on no hi ha les mesures nutritives comunes als països industrialitzats (una dieta variada, programes de fortificació i suplements dietètics). El bio-enriquiment mitjançant l’enginyeria genètica del contingut de Fe y Zn en arròs es una estratègia prometedora y pot resultar en un augmentant de les seves concentracions. Amb l’objectiu d’aconseguir arròs que acumuli nivells mes elevats de Fe y Zn he obtingut una població d’arròs transgènic mitjançant transformació combinatòria amb quatre gens implicats en l’absorció, transport, acumulació i biodisponibilitat d’aquestos dos minerals. Aquesta població combinatòria m’ha servit per a identificar els mecanismes antagònics i sinèrgics que son essencials per a controlar l’acumulació de Fe i Zn. / El arroz es un cultivo básico para más de 3 billones de personas. El arroz es una fuente pobre de hierro (Fe) y zinc (Zn) por lo cual las poblaciones pobres que dependen del arroz para su supervivencia, sufren de anemia por deficiencia de hierro (IDA) y zinc (ZnD). La desnutrición es un reto importante, especialmente en los países en desarrollo donde están ausentes las medidas que son comunes en los países industrializados (dieta variada, programas de fortificación y suplementos dietéticos). El bio-enriquecimiento mediante la ingeniería genética del contenido de Fe y Zn en arroz es una estrategia prometedora para aumentar sus concentraciones. Con el objetivo de producir granos con más alto nivel de Fe y Zn he obtenido una población de arroz transgénico mediante transformación combinatoria con cuatro genes implicados en la absorción, transporte, acumulación y biodisponibilidad de estos dos minerales. Esta población combinatoria me sirvió para identificar los mecanismos antagónicos y sinérgicos que son esenciales para controlar la acumulación de Fe y Zn. / Rice is staple crop for more than 3 billion people, since rice is poor source of iron (Fe) and zinc (Zn) world’s poorest people who depend on rice for their survival suffer from iron deficiency anaemia (IDA) and zinc deficiency (ZnD). Malnutrition is a significant challenge, particularly in the developing world where measures that are commonplace in industrialized countries (varied diet, fortification schemes, and dietary supplements) are largely absent. Biofortification of rice with Fe and Zn by genetic engineering is a promising strategy to increase Fe and Zn concentration. I established a combinatorial transgenic rice population by transformation with four genes involved in uptake, transport, accumulation and bioavailability of Fe and Zn, aiming to produce grains containing higher level of these two minerals. This combinatorial population helped to identify key antagonistic and synergistic mechanisms controlling Fe and Zn accumulation.

Construimos mediante ingeniería genética los diferentes vectores de expresión micobacteriano, a partir de los vectores parentales pMV261 y pMV361. Se desarrollaron 3 cepas BCG recombinantes expresando la proteína completa gp120 de la envuelta del VIH, cepa (HXBC2). Se evaluaron diferentes promotores de BCG para inducir la expresión de la proteína heteróloga del VIH. Inicialmente usamos el promotor hsp60 de BCG, un promotor fuerte que ha demostrado ser uno de los más eficaces y posteriormente, se probó con un promotor débil denominado alfa - antígeno. Una vez obtenidas las diferentes cepas BCG recombinantes se evaluó la expresión in vitro de la proteína mediante la técnica de Western blot. Posteriormente evaluamos in vivo la respuesta inmune celular específica, en un modelo animal murino. Se inmunizaron retones con las cepas BCGr: 222VIHA, BCGr: 223VIHA y virus vaccinia modificado cepa Ankara (VAM), expresando el inmunogeno del VIHA correspondiente a la proteína entera gag y envuelta del subtipo A predominante en África del este, más diferentes epítopos CTL de nef, pol. La respuesta específica de células T CD8+, se detectó por la tinción de los tetrámeros y por la tinción intracelular de IFN-alfa. Finalmente para superar las limitantes en la diferenciación de la inducción de citocinas entre BCG (cepa salvaje) y BCGr por la técnica de ELISPOT, evaluamos la expresión génica de IFN-alfa a partir de linfocitos de ratón, detectando el ARNm de esta citocina por la técnica de RT-PCR.Aportaciones de la tesis:- Diseño y desarrollo, por ingeniería genética y biología molecular de 3 cepas vacunales de BCG recombinante (BCGr:261VIH-1gp120, BCGr:222VIH-1gp120 y BCGr:223VIH-1gp120)- Estudio y descripción del reajuste genético que produce BCG cepa salvaje, cuando esta es transformada por el vector de expresión micobacteriano pMV261 que incluye clonado el fragmento de ADN que codífica la proteína del VIH-1 gp120 y que culmino con la deleción del mismo. (Este estudio se presento en un póster, en la Conferencia Mundial Sobre Vacunas VIH en Lausanne Suiza sept. 2004) - Los resultados de los ensayos preclínicos, para la evaluación de la inmunogenicidad de nuestras cepas vacunales, para inducir respuesta celular T CD8 específica frente al VIH-1 en ratones, evaluada por diferentes técnicas, proporcionan experiencias de utilidad en futuros ensayos referentes a las dosis, vías de administración y régimen empleado, así como en la selección de la técnica para detectar dicha respuesta inmune.

Crop productivity is limited by a number of important constraints that need to be addressed urgently in order to avoid an imminent humanitarian crisis. My thesis provides three diverse yet converging examples of biotechnological solutions that can deliver fundamental knowledge, tools and potential products in the form of improved/enhanced crop plants. I conclude my thesis by discussing the potential of biotechnology to address the MDGs. My key conclusion is that although biotechnology can contribute positively and substantially towards many of the MDGs, political expediency and an over-burdening regulatory system threaten to prevent those needing the technology from gaining access, i.e. impoverished subsistence farmers and their families in the developing world.