Flavonoids are chemically modified by glucosylation, hydroxylation, methylation, etc. During glucosylation, the sugar moiety from UDP-sugar is transferred to aglycone flavonoid substrates by glucosyltransferases (GTs). Grapefruit contains 5 different glucosyltransferases that demonstrate differences in not only substrate but also position specificity. Previous research obtained 3 putative 5’ grapefruit GT clones using SMART RACE RT-PCR with a degenerate gene specific primer based on a highly conserved sequence area in the Plant Secondary Product Glucosyltransferase box. The objective of this research was to use clone specific primers to obtain 3’ clones of the 3 previously mentioned 5’ clones as well as verify putative GT candidacy based on sequence data. Two of the 3 putative GT candidates were designated non-GTs following 3’end sequencing. During pursuit of sequence for the remaining 5’ clone, 1 full-length clone and 1 partial putative GT clone were obtained. To verify GT status, the clones must undergo expression/biochemical characterization.

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.

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.

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