title: Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.
abstract: Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform.
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France.
authors.forename: E
authors.initials: E
authors.lastname: Foureau
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France.
authors.forename: I
authors.initials: I
authors.lastname: Carqueijeiro
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France.
authors.forename: T
authors.initials: T
authors.lastname: Dugé de Bernonville
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France.
authors.forename: C
authors.initials: C
authors.lastname: Melin
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France.
authors.forename: F
authors.initials: F
authors.lastname: Lafontaine
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France.
authors.forename: S
authors.initials: S
authors.lastname: Besseau
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France.
authors.forename: A
authors.initials: A
authors.lastname: Lanoue
authors.affiliation: Université d'Angers, Groupe d'Etude des Interactions Hôte-Pathogène, UPRES EA 3142, Angers, France.
authors.forename: N
authors.initials: N
authors.lastname: Papon
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France.
authors.forename: A
authors.initials: A
authors.lastname: Oudin
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France.
authors.forename: G
authors.initials: G
authors.lastname: Glévarec
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France.
authors.forename: M
authors.initials: M
authors.lastname: Clastre
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France.
authors.forename: B
authors.initials: B
authors.lastname: St-Pierre
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France.
authors.forename: N
authors.initials: N
authors.lastname: Giglioli-Guivarc'h
authors.affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", Tours, France. Electronic address: vincent.courdavault@univ-tours.fr.
authors.forename: V
authors.initials: V
authors.lastname: Courdavault
mesh_terms: D053898:Biosynthetic Pathways; D030008:Catharanthus / Q000166:cytology / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D017343:Genes, Plant; D023281:Genomics / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D058615:Synthetic Biology / Q000379:methods*; D059467:Transcriptome; D014170:Transformation, Genetic; D015003:Yeasts / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
keywords: Biolistic; Candidate gene identification; Catharanthus roseus; Fluorescent protein fusions; Specialized metabolism; Subcellular localization; Synthetic biology; Virus-induced gene silencing; Yeast bioengineering