title: Expression of an evolved engineered variant of a bacterial glycine oxidase leads to glyphosate resistance in alfalfa.
abstract: The main strategy for resistance to the herbicide glyphosate in plants is the overexpression of an herbicide insensitive, bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). A glyphosate resistance strategy based on the ability to degrade the herbicide can be useful to reduce glyphosate phytotoxicity to the crops. Here we present the characterization of glyphosate resistance in transgenic alfalfa (Medicago sativa L.) expressing a plant-optimized variant of glycine oxidase (GO) from Bacillus subtilis, evolved in vitro by a protein engineering approach to efficiently degrade glyphosate. Two constructs were used, one with (GO(TP+)) and one without (GO(TP-)) the pea rbcS plastid transit peptide. Molecular and biochemical analyses confirmed the stable integration of the transgene and the correct localization of the plastid-imported GO protein. Transgenic alfalfa plants were tested for glyphosate resistance both in vitro and in vivo. Two GO(TP+) lines showed moderate resistance to the herbicide in both conditions. Optimization of expression of this GO variant may allow to attain sufficient field resistance to glyphosate herbicides, thus providing a resistance strategy based on herbicide degradation.
authors.affiliation: Dipartimento di Scienze Agrarie, Alimentari e Ambientali, University of Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy. Electronic address: alessandro.nicolia@gmail.com.
authors.forename: A
authors.initials: A
authors.lastname: Nicolia
authors.affiliation: Dipartimento di Scienze Agrarie, Alimentari e Ambientali, University of Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Ferradini
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, via J.H. Dunant 3, 21100 Varese, Italy; Centro Interuniversitario di Ricerca in Biotecnologie Proteiche The Protein Factory, Politecnico di Milano, ICRM CNR Milano and Università degli studi dell'Insubria, via Mancinelli 7, 20131 Milano, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Molla
authors.affiliation: Dipartimento di Scienze Agrarie, Alimentari e Ambientali, University of Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Biagetti
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, via J.H. Dunant 3, 21100 Varese, Italy; Centro Interuniversitario di Ricerca in Biotecnologie Proteiche The Protein Factory, Politecnico di Milano, ICRM CNR Milano and Università degli studi dell'Insubria, via Mancinelli 7, 20131 Milano, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Pollegioni
authors.affiliation: Dipartimento di Scienze Agrarie, Alimentari e Ambientali, University of Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Veronesi
authors.affiliation: Dipartimento di Scienze Agrarie, Alimentari e Ambientali, University of Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Rosellini
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D053209:Herbicide Resistance / Q000235:genetics*; D000455:Medicago sativa / Q000187:drug effects / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics; D015202:Protein Engineering; D000097797:Glyphosate
chemical_list: D000594:Amino Acid Oxidoreductases; D005998:Glycine; D000097797:Glyphosate; C115925:glycine oxidase
keywords: Glycine oxidase; Glyphosate; Herbicide resistance; Medicago sativa; Protein engineering