title: In vivo evolution of metabolic pathways by homeologous recombination in mitotic cells.
abstract: We describe a rapid and highly efficient method for the assembly, recombination, targeted chromosomal integration and regulatable expression of mosaic metabolic pathways by homeologous recombination in DNA repair deficient yeast cells. We have assembled and recombined 23kb pathways containing all the genes encoding enzymes for the production of flavonoids, a group of plant secondary metabolites of nutritional and agricultural value. The mosaic genes of the pathways resulted from pair-wise recombination of two nonidentical (homeologous) wild-type genes. The recombination events occurred simultaneously in the cell. Correctly assembled mosaic gene clusters could only be observed in DNA repair deficient strains. Thus, libraries of intragenic mosaic pathways were generated. Randomly isolated clones were screened for their ability to produce flavonoids such as kaempferol, phloretin and galangin. Thus, the functionality of the recombinant pathways was proven. Additionally, significant higher concentrations of metabolites such as naringenin, pinocembrin and dihydrokaempferol were detected. Further analysis also revealed the production of different aromatic compounds such as styrene, hydroxystyrene, phloretic acid and other molecules. We show that the in vivo homeologous recombination strategy can generates libraries of intragenic mosaic pathways producing a high diversity of phenylpropanoid compounds.
authors.affiliation: EVIAGENICS, 1 Mail du Professeur Georges Mathé, 94800 Villejuif, France. Electronic address: luque@eviagenics.com.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Luque
authors.affiliation: EVIAGENICS, 1 Mail du Professeur Georges Mathé, 94800 Villejuif, France.
authors.forename: Sarra C
authors.initials: SC
authors.lastname: Sebai
authors.affiliation: ZEA-3/BioSpec, Research Centre Jülich GmbH, 52425 Jülich, Germany.
authors.forename: Beatrix
authors.initials: B
authors.lastname: Santiago-Schübel
authors.affiliation: EVIAGENICS, 1 Mail du Professeur Georges Mathé, 94800 Villejuif, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Le Coz
authors.affiliation: EVIAGENICS, 1 Mail du Professeur Georges Mathé, 94800 Villejuif, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Jenot
authors.affiliation: EVIAGENICS, 1 Mail du Professeur Georges Mathé, 94800 Villejuif, France.
authors.forename: Odile
authors.initials: O
authors.lastname: Ramaen
authors.affiliation: EVIAGENICS, 1 Mail du Professeur Georges Mathé, 94800 Villejuif, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Sauveplane
authors.affiliation: EVIAGENICS, 1 Mail du Professeur Georges Mathé, 94800 Villejuif, France.
authors.forename: Rudy
authors.initials: R
authors.lastname: Pandjaitan
mesh_terms: D019020:Directed Molecular Evolution / Q000379:methods*; D059765:Homologous Recombination; D060847:Metabolic Engineering / Q000379:methods*; D008938:Mitosis / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
keywords: DNA repair deficiency; Flavonoids; Heterologous expression; Homeologous/homologous recombination; In vivo evolution; Metabolic reconstitution; Mosaic pathways; Phenylpropanoids; Yeast