Flavonoids are a class of plant secondary metabolites that fulfill many functions in planta and also benefit human health. Glucosylation of flavonoids, catalyzed by glucosyltransferases (GTs), help flavonoids perform their important roles. Citrus paradisi (grapefruit) is known to be active in flavonoid metabolism and possesses a variety of secondary product GTs. This research is designed to test the hypothesis that PGT10 is a flavonoid glucosyltransferase. PGT10 was cloned, expressed, purified, and tested for GT activity with a variety of phenolic substrates. Efforts to identify the reaction catalyzed by PGT10 continue. A second project was designed to test the hypothesis that multiple bands obtained by RT-PCR of PGT 5/6 found in root cDNA represented a set of homologous genes. Hypothesis has been confirmed by the homologous fragments found. Additional structural information is presented.

Flavonoids are plant secondary metabolites well known for many key roles in the life cycle of plants. They also can affect human health. Citrus paradisi is known to produce several glucosylated flavonoids and these compounds are glucosylated by enzymes known as glucosyltransferases (GTs). The focus of this research was to optimize the heterologous expression, enrichment, and biochemical characterization of grapefruit putative GT protein, PGT2, and to test the hypothesis that PGT2 is a flavonoid GT. Results showed detectable amounts of activity with quercetin, a flavonol; however, activity was lower than what would be expected if this enzyme were a flavonol-specific GT. In an additional aspect of this study, bioinformatics were used to test the hypothesis that additional putative GT clones could be identified using the harvEST database.

Flavonoids are secondary metabolites that have significant roles in plant defense and human nutrition. Glucosyltransferases (GTs) transfer sugars from high energy sugar donors to other substrates. Several different kinds of flavonoid GTs exist in the tissues of grapefruit making it a model plant for studying their structure and function. The goal of this investigation is to determine the expression patterns of 7 putative secondary product GTs during grapefruit growth and development by quantifying mRNA expression levels in the roots, stems, leaves, and flowers. This research was designed to test the hypothesis that these 7 GT's are expressed constitutively. Alternatively, one or more could be expressed in a tissue-specific manner and/or developmentally regulated. Six growth stages were defined. Findings show that there were variable degrees of PGT expression. Therefore, results were more consistent with the alternative hypothesis that putative secondary product GT expression was tissue specific and/or developmentally regulated.

The general objective of this study was to analyse freeze-drying (FD) and spray- drying (SD) and to select the best process to obtain a grapefruit powder of high nutritional, functional and sensorial quality. With this objective in mind, the optimization of each dehydration process was carried out using the response surface methodology. As variables of the processes, in both cases, the concentration of gum arabic (GA) and bamboo fibre (BF), added as carriers, were considered, as well as the feed inlet moisture (Xw) in the case of FD or air inlet temperature (T) in SD. The properties of the obtained products analyzed were the water content, hygroscopicity, porosity, color, bioactive compounds (vitamin C, total carotenoids, total phenols) and antioxidant activity (AOA). In addition, the drying yield and product yield of SD were considered. The obtained results, duly validated, allow to propose for FD, the addition of 4.2g GA and 0.58g BF per 100g of grapefruit pulp and bringing the sample to 90g water/100g feed mixture. In the case of SD, the best product is obtained with T in the equipment is of 120 °C and the addition of 4g AG and 2g BF/100g liquidized grapefruit. The comparison of both products allows us to propose FD as being a better technique than spray-drying. In addition, the powder yield of FD is much higher compared to SD and far fewer by-products are generated. A specific study was carried out to confirm the encapsulating power of the two solutes used. The results obtained confirm the benefit of the joint addition of GA and BF, especially against the spray-drying temperature, offering a greater protection against the degradation of bioactive compounds and AOA, than when solutes are added separately. The characterization of the different bioactive compounds and their correlation with the AOA carried showed that the phenolic compounds contribute significantly to the free radical scavenging activity and the inhibition of the discoloration of ß -carotene, while ascorbic acid and ¿- tocopherol contribute to an increase in reducing power. In addition, the stability of the optimized powders during storage at 4 and 20 °C and different surrounding relative humidities has been studied. The modeling of the water sorption data together with the variation in the glass transition temperature (Tg) that takes place in line with the water content of the samples permits the determination of the critical values of water content and water activity that ensure the glassy state of the powdered product. In this sense, what is recommended when storing the powders is to maintain the products under refrigeration and with a surrounding relative humidity in the order of 10%. As soon as the grapefruit powder begins the Tg, the degradation of the bioactive compounds begins, with carotenoids being the most sensitive. In less restrictive storage conditions than those mentioned above, in just one month of storage there are already significant losses in the studied compounds, fewer in the FD product than in the SD. In order to detect any problems of caking in the free-flowing powders or changes in color, a larger quantity of the product must have changed to the rubbery state. In this sense, for the purposes of relating the loss in bioactive compounds, the change in the mechanical properties and the color change with the glass transition, the onset temperature, the midpoint or the end point of the Tg, respectively, should be taken into consideration. Finally, a sensory evaluation of the rehydrated powdered products was carried out in order to evaluate consumer acceptance of the juices obtained and their probability of purchase. In general, grapefruit juice, even the natural or commercial ones, did not enjoy a good level of consumer acceptance due to its astringency and high acidity. The penalty analysis showed that if the sweetness of the samples is improved and their astringency is reduced, their level of consumer acceptance will rise. / El objetivo general de este estudio fue seleccionar el mejor proceso entre la liofilización y la atomización para la obtención de pomelo en polvo de alta calidad nutritiva, funcional y sensorial. Para ello se llevó a cabo la optimización de ambos procesos, usando la metodología de superficie de respuesta. Como variables independientes se consideraron, en ambos casos, la cantidad de goma arábiga (GA) y de fibra de bambú (FB) incorporadas, y la cantidad de agua de la muestra (Xw) en la liofilización o la temperatura de atomización (T). Las propiedades analizadas a los productos obtenidos fueron la humedad, higroscopicidad, porosidad, color, vitamina C, carotenoides totales, fenoles totales y la actividad antioxidante (AOA). Además se consideró el rendimiento en producto de la atomización. Los resultados obtenidos, debidamente validados, permiten proponer, para la liofilización, la adición de 4.2g de GA y 0.58g de FB por cada 100 g de pomelo triturado y una Xw de 90 gagua/100gmezcla. En el caso de la atomización, el mejor producto se obtiene cuando la T es de 120 °C y al pomelo licuado se añaden 4g GA y 2g de FB por cada 100g. Al comparar ambos productos se propone a la liofilización como una mejor tecnología de secado que la atomización. Además, se llevó a cabo un estudio para confirmar el poder encapsulante de los dos solutos utilizados. Los resultados confirmaron el beneficio de la adición conjunta de GA y FB especialmente frente a la temperatura de atomización, ofreciendo una mayor protección de la degradación de los compuestos bioactivos y de la AOA que cuando se añaden por separado. Por su parte, la caracterización de los diferentes compuestos bioactivos y su correlación con la AOA realizada en esta parte del estudio, mostró que los compuestos fenólicos contribuyen de manera significativa a la actividad captadora de radicales libres y a la inhibición de la decoloración de ß-caroteno, mientras que el ácido ascórbico y el ¿-tocoferol contribuyen a incrementan el poder reductor. Además se estudió la estabilidad de los polvos optimizados durante el almacenamiento, a 4 y a 20 °C y a diferentes humedades relativas (HR). La modelización conjunta de los datos de sorción de agua y de variación de la temperatura de transición vítrea (Tg) con la humedad de las muestras permite establecer los valores críticos de humedad y actividad del agua que aseguran el estado vítreo del producto en polvo. En este sentido, para su almacenamiento se recomienda la refrigeración y mantener la HR del entorno del orden del 10 %. En cuanto en el pomelo en polvo se inicia la Tg, comienza la degradación de los compuestos bioactivos, siendo los carotenoides los más sensibles. En condiciones de almacenamiento menos restrictivas a las comentadas, en tan sólo un mes de almacenamiento ya se observan pérdidas importantes de los compuestos estudiados, que son menores en el producto liofilizado que en el atomizado. Por otra parte, para que en el almacenamiento empiecen a detectarse problemas de apelmazamiento del polvo suelto o cambios de color, es necesario que una mayor cantidad de producto haya pasado a estado gomoso. En este sentido, para el estudio de la pérdida de compuestos bioactivos, del cambio en las propiedades mecánicas y del cambio de color se recomienda considerar la temperatura inicial, la del punto medio o la del punto final de la Tg, respectivamente. Finalmente se realizó un análisis sensorial de los productos en polvo rehidratados para evaluar el grado de aceptación de los zumos así obtenidos y su probabilidad de compra. En general el zumo de pomelo, no mostró una buena aceptación por parte del consumidor debido a su astringencia y elevada acidez. El análisis de penalización mostró que si se mejora el dulzor de las muestras y se disminuye su astringencia podrían mejorar los productos y cambiar su grado de aceptación. / L'objectiu general d'este estudi va ser seleccionar el millor procés entre la liofilització (LIO) i l'atomització (ATO) per a l'obtenció de pomelo en pols d'alta qualitat nutritiva, funcional i sensorial. Amb este objectiu es va dur a terme l'optimització de cada procés de deshidratació usant la metodologia de superfície de resposta. Com a variables dels processos es van considerar, en ambdós casos, la quantitat de goma aràbiga (GA) i de fibra de bambú (FB) incorporades com coadjuvants dels processos, així com la quantitat d'aigua de la mostra en el cas de la LIO o la temperatura d'ATO. Les propietats analitzades als productes obtinguts van ser la humitat, higroscopicidad, porositat, color, compostos bioactivos (vitamina C, carotenoides totals, fenols totals) i l'AOA. A més es va considerar el rendiment en producte de l'atomització. Els resultats obtinguts, degudament validats, permeten proposar, per a la LIO, la incorporació de 4.2g de GA i 0.58g de FB per cada 100g de pomelo triturat i portar a la mostra abans del seu processat fins una humitat de 90g agua/100g mezcla. En el cas de l'ATO, el millor producte s'obté quan la T en l'equip és de 120 °C i al liquat de pomelo s'afigen 4g de GA i 2g de FB per cada 100g. La comparació d'ambdós productes permet proposar a la LIO com una millor tecnologia d'assecat que l'ATO. A més el rendiment en producte de la LIO és molt major que el de l'ATO i els subproductes generats molt menors. D'altra banda, es va dur a terme un estudi específic per a confirmar el poder encapsulant dels dos soluts utilitzats. Els resultats permeten confirmar el benefici de l'addició conjunta de GA i FB especialment enfront de la temperatura d'ATO, que oferixen una major protecció de la degradació de compostos bioactivos i AOA que quan s'afigen per separat. La caracterització dels diferents compostos bioactivos i la seua correlació amb l'AOA realitzada en esta part de l'estudi, va mostrar que els compostos fenòlics contribuïxen de manera significativa a l'activitat captadora de radicals lliures i a la inhibició de la decoloració del ¿-caroteno, mentres que l'àcid ascòrbic i el ¿-tocoferol contribuïxen a incrementen el poder reductor. A més s'ha estudiat l'estabilitat de les pols optimitzats durant l'emmagatzemament, a 4 i 20 °C i a diferents humitats relatives (HR) de l'entorn. La modelització de les dades de sorción d'aigua i de variació de la temperatura de transició vítria (Tg) amb la humitat de les mostres permet establir els valors crítics d'humitat i activitat de l'aigua que asseguren l'estat vitri del producte en pols. En este sentit, per al seu emmagatzemament es recomana la refrigeració i mantindre la HR de l'entorn de l'orde del 10 %. En quant en el pomelo en pols s'inicia la Tg, comença la degradació dels compostos bioactivos. En condicions menys restrictives a les comentades, en tan sols un mes d'emmagatzemament ja s'observen pèrdues importants dels compostos estudiats, que són menors en el producte LIO que en l'ATO. D'altra banda, perquè en l'emmagatzemament comencen a detectar-se problemes d'atapeïment de la pols solta o canvis de color, és necessari que una major quantitat de producte haja passat a estat gomós. En este sentit, per a l'estudi de la pèrdua de compostos bioactivos, del canvi en les propietats mecàniques i del canvi de color es recomana considerar la temperatura inicial, la del punt mitjà o la del punt final de la Tg, respectivament. Finalment es va realitzar una anàlisi sensorial dels productes en pols rehidratats per a avaluar el grau d'acceptació dels sucs així obtinguts i la seua probabilitat de compra. En general el suc de pomelo, inclús el natural o el comercial, no va mostrar una bona acceptació per part del consumidor a causa de la seua astringència i elevada acidesa. L'anàlisi de penalització va mostrar que si es millora la dolçor de les mostres i es disminuïx la seua astringència podrien millorar els produc / Agudelo Sterling, CM. (2017). SELECCIÓN DEL MEJOR PROCESO PARA LA OBTENCIÓN DE POMELO EN POLVO (Citrus Paradisi) DE ALTA CALIDAD NUTRITIVA, FUNCIONAL Y SENSORIAL [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/80695

Les furocoumarines sont des composés phénoliques impliqués dans la défense contre les herbivores. Ces molécules sont majoritairement décrites dans quatre familles botaniques, notamment les Rutaceae, dont font partie les agrumes. Ces molécules sont phototoxiques ce qui peut poser des problèmes pour leur utilisation comme par exemple en cosmétique ou en phytothérapie. D’autre part, en cas d’ingestion par exemple via la consommation de jus de certains agrumes, elles ont responsables de l’inhibition d’enzymes de détoxication comme le CYP3A4 humain. Cela peut conduire à des surdosages médicamenteux connus sous le nom d’Effet Pomelo. Ce travail de thèse a consisté à réfléchir et à développer, des outils qui permettront de générer de manière ciblée des variétés de pomelo qui ne produisent plus de furocoumarines. Nous avons abordé l’ensemble des étapes essentielles pour la mise en place d’une stratégie global : i) des méthodes reproductibles ont été développées pour la production de protoplastes et de cultures cellulaires de pomelo Star Ruby ; ii) des conditions de transformation de protoplastes par électroporation ont également été mises au point ; iii) finalement, pour inhiber de manière spécifique la voie de biosynthèse des furocoumarines, nous avons choisi de mettre en œuvre une approche d’édition de génome en utilisant une méthodologie CRISPR/Cas9. La mise au point de la méthode a été réalisée avec un gène codant pour une umbelliferone 6-dimethylallyl transférase. Les résultats obtenus indiquent que la stratégie est envisageable. Pour renforcer la stratégie CRISPR/Cas9, nous avons mis en œuvre une démarche d’identification de gènes cibles additionnels. En utilisant une approche de data mining de bases de données génomiques et transcriptomiques nous avons identifié 18 séquences candidates, potentiellement impliquées dans la voie de biosynthèse des furocoumarines. L’expression hétérologue des protéines correspondantes et leur caractérisation fonctionnelle a permis de montrer que CYP706J12 est en mesure de métaboliser l’hérniarine, une coumarine. Ce résultat apporte des éléments pour émettre des hypothèses sur l’évolution convergente de la synthèse des coumarines et des furocoumarines chez les végétaux supérieurs. / Furanocoumarins are phenolic compounds involved in defense against herbivores. These molecules are mainly described in four botanical families. Rutaceae, one of those families, includes Citrus species. Furanocoumarins are phototoxic compounds, which can be problematic for their use in cosmetics or in phytotherapy. Furanocoumarin ingestion via citrus juice consumption, may inhibit human enzymes of detoxification, such as human CYP3A4. This can lead to drug overdoses known as the “Grapefruit Juice Effect”. This work consisted in the development of tools that will allow to generate new varieties of pomelo that no longer produce furanocoumarins by targeted genome edition. We have covered the essential steps for the implementation of a global strategy: i) reproducible methods have been developed for the production of protoplasts and cell cultures of Star Ruby grapefruit; ii) conditions for protoplast transformation by electroporation have also been developed; iii) finally, to specifically inhibit the furanocoumarin biosynthetic pathway, we chose to implement a genome editing approach using a CRISPR / Cas9 methodology. The development of the method was carried out with a gene encoding umbelliferon 6-dimethylallyltransferase. The results obtained indicate that the strategy is feasible. To strengthen the CRISPR / Cas9 strategy, we implemented a method to identify additional target genes. Using a data mining approach of available genomic and transcriptomic databases we identified 18 candidate sequences potentially involved in the furanocoumarin biosynthetic pathway. Heterologous expression of the corresponding proteins and their functional characterization made it possible to show that CYP706J12 is able to metabolize herniarin (a coumarin). This result provides elements to hypothesize about the convergent evolution of coumarin and furanocoumarin synthesis in higher plants.

Citrus and other fruits produce secondary metabolites that are synthesized, regulated, and modified in part by a class of enzymes called glycosyltransferases. This class of enzymes is of substantial interest to this lab due to their unique structural and functional properties. Glycosides of flavonoids produced by glycosyltransferases have emerged in recent years as a critical part of plant metabolism, thus impacting every aspect of their growth, cultivation, production, and utilization. One such glycosyltransferase, found in Duncan Grapefruits (Citrus paradisi), was previously identified, recombinantly expressed, and shown through biochemical characterization to exclusively glycosylate the flavonol class of flavonoids. The structural basis that accounts for a glycosyltransferase's selectivity has been determined by protein crystallization in other labs, yet no structural basis currently exists for the specificity exhibited by this flavonol-specific glycosyltransferase. Currently, the WT enzyme and two mutants were expressed in E. coli, where they underwent site-directed mutagenesis to insert thrombin cleavage tags for removal of protein purification vectors, with the goal of transforming into yeast for adequate protein production. Subsequent purification and crystallization screens will allow for formation and acquisition of glycosyltransferase crystals, whose x-ray diffraction patterns will be decoded, thus revealing the enzyme's complete structure. We hypothesize that obtaining a crystal structure for this enzyme will illuminate the structural basis of its specificity. Additionally, we hypothesize that a thrombin- cleavage gene vector inserted for removal of purification tags will have no impact on enzyme activity or specificity.

Flavonoids are the C-15 phenolic compounds containing two phenyl rings and a heterocyclic ring. The majority of the flavonoids accumulated in grapefruit are flavonol, flavanone, flavone, dihydroflavonol, and chalcone glycosides. Most flavonoids are present in glucosylated form and the glucosylation is mediated by a class of enzymes called glucosyltransferases that transfer glucose from a high energy sugar donor to the acceptor aglycone at a particular position. A clone encoding a flavonol-specific 3-O-glucosyltransferase (Cp-3-O-GT) from Citrus paradisi has been previously characterized in our lab. The study of structure and function of flavonoid GTs is an important aspect of our research that contributes to the synthesis of novel glucosides by changing the glucosylation patterns of GTs. Our study focuses on the structural and functional analysis of Cp-3-O-GT through site directed mutation and analysis of mutated enzyme in terms of substrate specificity and regiospecificity. Multiple sequence alignment and homology modeling was used to identify candidate areas for mutation. For this study, Cp-3-O-GT was modeled with a flavonoid 3- O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GT – P145T mutant to test the hypothesis that that mutation of key amino acid residues (proline) in Cp-3-O-GT by position specific amino acids of VvGT (threonine) could alter substrate specificity or regiospecificity of Cp-3-O-GT. Initial screening results suggested that the mutant P145T glucosylates flavanones and flavones in addition to flavonols. This is significant because flavanones and flavonols do not contain a 3-OH group for glucosylation. HPLC was performed to identify the reaction products. Early results indicate that the P145T mutant glucosylates naringenin at 7-OH position forming naringenin-7-O-glucoside and this is being confirmed. Product identification with other substrates is also being conducted. Results are being used to revisit and refine the structure model.

Glucosyltransferases (GTs) are the important group of enzymes which facilitates the incorporation of UDPactivated glucose to a corresponding acceptor molecule through glucosylation. Glucosylation is a common alteration reaction in plant metabolism and is regularly associated with the production of secondary metabolites. Glucosylation serves a number of roles within metabolism including: stabilizing structures, affecting solubility, transport, and regulating the bioavailability of the compounds for other metabolic processes. GTs involved in secondary metabolism share a conserved 44 amino acid residue motif (60–80% identity) known as the plant secondary product glucosyltransferase (PSPG) box, which has been demonstrated to include the UDP-sugar binding moiety. Among the secondary metabolites, flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant Flavonol- 3-O- Glucosyltransferases proteins using site-directed mutational analysis and testing the effect of each mutation on substrate specificity and kinetic properties of the enzyme.

Glucosyltransferases (GTs) are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi has strict substrate and regiospecificity (Cp3OGT). The amino acid sequence of Cp3OGT was aligned with a purported anthocyanin GT from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling to identify candidate regions followed by site directed mutagenesis, three double mutations of Cp3OGT were made. Biochemical analysis of the three mutant proteins was performed. S20G+T21S protein retained activity similar to the wildtype (WT- Kmapp-80 µM; Vmax = 16.5 pkat/µg, Mutant- Kmapp-83 µM; Vmax -11 pkat/µg) but the mutant was more thermostable compared to the WT and this mutation broadened its substrate acceptance to include the flavanone, naringenin. S290C+S319A mutant protein retained 40% activity relative to wildtype, had an optimum pH shift, but had no change in substrate specificity (Kmapp-18 µM; Vmax-0.5 pkat/µg). H154Y+Q87I protein was inactive with every class of flavonoid tested. Product identification revealed that the S20G+T21S mutant protein widened the substrate and regio-specificity of CP3OGT. Docking analysis revealed that H154 and Q87 could be involved in orienting the ligand molecules within the acceptor binding site. H363, S20, and S150 were also found to make close contact with the 7-OH, 4-OH and 3’-OH groups, respectively.

No description available.