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Luque A.,EVIAGENICS | Sebai S.C.,EVIAGENICS | Santiago-Schubel B.,Jülich Research Center | Le Coz Y.,EVIAGENICS | And 4 more authors.
Metabolic Engineering | Year: 2014

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 23. kb 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. © 2014.


Escoffre J.-M.,CNRS Institute of Pharmacology and Structural Biology | Escoffre J.-M.,Toulouse 1 University Capitole | Escoffre J.-M.,University Utrecht | Bellard E.,CNRS Institute of Pharmacology and Structural Biology | And 9 more authors.
Biochimica et Biophysica Acta - Biomembranes | Year: 2014

Membrane electropermeabilization relies on the transient permeabilization of the plasma membrane of cells submitted to electric pulses. This method is widely used in cell biology and medicine due to its efficiency to transfer molecules while limiting loss of cell viability. However, very little is known about the consequences of membrane electropermeabilization at the molecular and cellular levels. Progress in the knowledge of the involved mechanisms is a biophysical challenge. As a transient loss of membrane cohesion is associated with membrane permeabilization, our main objective was to detect and visualize at the single-cell level the incidence of phospholipid scrambling and changes in membrane order. We performed studies using fluorescence microscopy with C6-NBD-PC and FM1-43 to monitor phospholipid scrambling and membrane order of mammalian cells. Millisecond permeabilizing pulses induced membrane disorganization by increasing the translocation of phosphatidylcholines according to an ATP-independent process. The pulses induced the formation of long-lived permeant structures that were present during membrane resealing, but were not associated with phosphatidylcholine internalization. These pulses resulted in a rapid phospholipid flip/flop within less than 1 s and were exclusively restricted to the regions of the permeabilized membrane. Under such electrical conditions, phosphatidylserine externalization was not detected. Moreover, this electrically-mediated membrane disorganization was not correlated with loss of cell viability. Our results could support the existence of direct interactions between the movement of membrane zwitterionic phospholipids and the electric field. © 2014 Elsevier B.V.


Luque A.,EVIAGENICS | Sebai S.C.,EVIAGENICS | Sauveplane V.,EVIAGENICS | Ramaen O.,EVIAGENICS | Pandjaitan R.,EVIAGENICS
Bioengineered Bugs | Year: 2014

In our recent article “In vivo evolution of metabolic pathways by homeologous recombination in mitotic cells” we proposed a useful alternative to directed evolution methods that permits the generation of yeast cell libraries containing recombinant metabolic pathways from counterpart genes. The methodology was applied to generate single mosaic genes and intragenic mosaic pathways. We used flavonoid metabolism genes as a working model to assembly and express evolved pathways in DNA repair deficient cells. The present commentary revises the principles of gene and pathway mosaicism and explores the scope and perspectives of our results as an additional tool for synthetic biology. © Alejandro Luque, Sarra C Sebai, Vincent Sauveplane, Odile Ramaen, and Rudy Pandjaitan.


Patent
Eviagenics | Date: 2013-10-09

The invention relates to a method for generating a gene mosaic by somatic in vivo recombination, comprisinga) in a single step procedure(i) transforming a cell with at least one gene A having a sequence homology of less than 99.5% to another gene to be recombined that is an integral part of the cell genome or presented in the framework of a genetic construct,(ii) recombining said genes,(iii) generating a gene mosaic of the genes at an integration site of a target genome, wherein said at least one gene A has a single flanking target sequence either at the 5 end or 3 end anchoring to the 5or 3end of said integration site, andb) selecting clones comprising the gene mosaic, as well as a method of producing a diversity of gene mosaics and gene assembly.


Patent
Eviagenics | Date: 2012-10-12

The invention relates to a method for generating a gene mosaic by somatic in vivo recombination, comprising a) in a single step procedure (i) transforming a cell with at least one gene A having a sequence homology of less than 99.5% to another gene to be recombined that is an integral part of the cell genome or presented in the framework of a genetic construct, (ii) recombining said genes, (iii) generating a gene mosaic of the genes at an integration site of a target genome, wherein said at least one gene A has a single flanking target sequence either at the 5 end or 3 end anchoring to the 5 or 3 end of said integration site, and b) selecting clones comprising the gene mosaic, wherein said cell is a eukaryotic strain with a knock-out of at least one DNA repair gene. The invention further refers to a method of producing a diversity of gene mosaics and gene assembly.


Patent
Eviagenics | Date: 2011-04-08

The invention relates to a method for generating a gene mosaic by somatic in vivo recombination, comprising: e) in a single step procedure (vii) transforming a cell with at least one gene A having a sequence homology of less than 99.5% to another gene to be recombined that is an integral part of the cell genome or presented in the framework of a genetic construct, (viii) recombining said genes, (ix) generating a gene mosaic of the genes at an integration site of a target genome, wherein said at least one gene A has a single flanking target sequence either at the 5 end or 3 end anchoring to the 5 or 3 end of said integration site, and f) selecting clones comprising the gene mosaic, as well as a method of producing a diversity of gene mosaics and gene assembly.


The invention relates to a cell comprising heterologous polynucleotides encoding a multienzyme complex involved in the metabolic pathway of phenylpropanoids and biosynthesis of a vanilloid or a hydroxybenzaldehyde precursor thereof, which multienzyme complex comprises enzymes for the biosynthesis of coumaric acid and a crotonase.


PubMed | CNRS Institute of Pharmacology and Structural Biology, Matwin Institute Bergonie and EVIAGENICS
Type: Journal Article | Journal: Biochimica et biophysica acta | Year: 2014

Membrane electropermeabilization relies on the transient permeabilization of the plasma membrane of cells submitted to electric pulses. This method is widely used in cell biology and medicine due to its efficiency to transfer molecules while limiting loss of cell viability. However, very little is known about the consequences of membrane electropermeabilization at the molecular and cellular levels. Progress in the knowledge of the involved mechanisms is a biophysical challenge. As a transient loss of membrane cohesion is associated with membrane permeabilization, our main objective was to detect and visualize at the single-cell level the incidence of phospholipid scrambling and changes in membrane order. We performed studies using fluorescence microscopy with C6-NBD-PC and FM1-43 to monitor phospholipid scrambling and membrane order of mammalian cells. Millisecond permeabilizing pulses induced membrane disorganization by increasing the translocation of phosphatidylcholines according to an ATP-independent process. The pulses induced the formation of long-lived permeant structures that were present during membrane resealing, but were not associated with phosphatidylcholine internalization. These pulses resulted in a rapid phospholipid flip/flop within less than 1s and were exclusively restricted to the regions of the permeabilized membrane. Under such electrical conditions, phosphatidylserine externalization was not detected. Moreover, this electrically-mediated membrane disorganization was not correlated with loss of cell viability. Our results could support the existence of direct interactions between the movement of membrane zwitterionic phospholipids and the electric field.


Patent
Eviagenics | Date: 2012-06-20

The invention relates to a method for metabolic evolution of a variant of a natural small aromatic molecule product of a metabolic pathway, by somatic in vivo assembly and recombination of said metabolic pathway employing a gene mosaic of at least one gene A, which comprises a) in a single step procedure (i) transforming a cell with at least one gene A having a sequence homology of less than 99.5% to another gene to be recombined that is an integral part of the cell genome or presented in the framework of a genetic construct, (ii) recombining said genes, (iii) generating a gene mosaic of the genes at an integration site of a target genome, wherein said at least one gene A has a single flanking target sequence either at the 5 end or 3 end anchoring to the 5 or 3 end of said integration site, (iv) recombining eventual further genes of said metabolic pathway, and b) selecting clones comprising said gene mosaic and said eventual further genes capable of expressing said variant, methods of preparing a library of cells producing variants of natural small aromatic molecule products of a metabolic pathway, the libraries so produced and used to prepare said variants.


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