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1.
Bioengineered ; 5(6): 347-56, 2014.
Article in English | MEDLINE | ID: mdl-25482082

ABSTRACT

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.


Subject(s)
Directed Molecular Evolution/methods , Homologous Recombination , Metabolic Engineering/methods , Mitosis/genetics , Saccharomyces cerevisiae
2.
Metab Eng ; 23: 123-35, 2014 May.
Article in English | MEDLINE | ID: mdl-24685654

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.


Subject(s)
Directed Molecular Evolution/methods , Homologous Recombination , Metabolic Engineering/methods , Mitosis/genetics , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
3.
Biochim Biophys Acta ; 1838(7): 1701-9, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24583083

ABSTRACT

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.


Subject(s)
Cell Membrane/metabolism , Phospholipids/metabolism , Adenosine Triphosphate/metabolism , Animals , CHO Cells , Cell Line , Cell Membrane Permeability , Cell Survival/physiology , Cricetulus , Electroporation/methods , Phosphatidylcholines/metabolism
5.
Langmuir ; 26(17): 14135-41, 2010 Sep 07.
Article in English | MEDLINE | ID: mdl-20704336

ABSTRACT

Membrane permeabilization is achieved via numerous techniques involving the use of molecular agents such as peptides used in antimicrobial therapy. Although high efficiency is reached, the permeabilization mechanism remains global with a noticeable lack of control. To achieve localized control and more gradual increase in membrane perturbation, we have developed hydrophobically modified poly(acrylic acid) amphiphilic copolymers with light-responsive azobenzene hydrophobic moieties. We present evidence for light triggered membrane permeabilization in the presence azobenzene-modified polymers (AMPs). Exposure to UV or blue light reversibly switches the polarity of the azobenzene (cis-trans isomerization) in AMPs, hence controlling AMP-loaded lipid vesicles permeabilization via in situ activation. Release of encapsulated probes was studied by microscopy on isolated AMP-loaded giant unilamellar vesicles (pol-GUVs). We show that in pH and ionic strength conditions that are biologically relevant pol-GUVs are kept impermeable when they contain predominantly cis-AMPs but become leaky with no membrane breakage upon exposure to blue light due to AMPs switch to a trans-apolar state. In addition, we show that AMPs induce destabilization of plasma membranes when added to mammal cells in their trans-apolar state, with no loss of cell viability. These features make AMPs promising tools for remote control of cell membrane permeabilization in mild conditions.


Subject(s)
Acrylic Resins/chemistry , Cell Membrane Permeability , Light , Membrane Lipids/chemistry , Acrylic Resins/chemical synthesis , Animals , Azo Compounds/chemistry , COS Cells , Cell Survival , Cells, Cultured , Chlorocebus aethiops , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Molecular Structure , Osmolar Concentration , Surface Properties
6.
Philos Trans A Math Phys Eng Sci ; 364(1847): 2597-614, 2006 Oct 15.
Article in English | MEDLINE | ID: mdl-16973478

ABSTRACT

Drug molecules must cross multiple cell membrane barriers to reach their site of action. We present evidence that one of the largest classes of pharmaceutical drug molecules, the cationic amphiphilic drugs (CADs), does so via a catalytic reaction that degrades the phospholipid fabric of the membrane. We find that CADs partition rapidly to the polar-apolar region of the membrane. At physiological pH, the protonated groups on the CAD catalyse the acid hydrolysis of the ester linkage present in the phospholipid chains, producing a fatty acid and a single-chain lipid. The single-chain lipids rapidly destabilize the membrane, causing membranous fragments to separate and diffuse away from the host. These membrane fragments carry the drug molecules with them. The entire process, from drug adsorption to drug release within micelles, occurs on a time-scale of seconds, compatible with in vivo drug diffusion rates. Given the rate at which the reaction occurs, it is probable that this process is a significant mechanism for drug transport.


Subject(s)
Lipid Bilayers/metabolism , Surface-Active Agents/pharmacokinetics , Biological Transport, Active , Cations , Diffusion , Drug Delivery Systems , In Vitro Techniques , Lipid Bilayers/chemistry , Liquid Crystals , Magnetic Resonance Spectroscopy , Microscopy, Fluorescence , Models, Biological , Phospholipids/chemistry , Phospholipids/metabolism , Scattering, Radiation , Surface-Active Agents/chemistry , X-Rays
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