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Biomedical subjects

Denis Pompon

Publications and source records attributed to Denis Pompon.

7 recordsLinked to original sources
PubMed2003

Total biosynthesis of hydrocortisone from a simple carbon source in yeast.

We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.

Animals↗
PubMed2003

Critical role of the plasma membrane for expression of mammalian mitochondrial side chain cleavage activity in yeast.

Engineered yeast cells efficiently convert ergosta-5-eneol to pregnenolone and progesterone provided that endogenous pregnenolone acetylase activity is disrupted and that heterologous sterol delta7-reductase, cytochrome P450 side chain cleavage (CYP11A1) and 3beta hydroxysteroid dehydrogenase/isomerase (3beta-HSD) activities are present. CYP11A1 activity requires the expression of the mammalian NADPH-adrenodoxin reductase (Adrp) and adrenodoxin (Adxp) proteins as electron carriers. Several parameters modulate this artificial metabolic pathway: the effects of steroid products; the availability and delivery of the ergosta-5-eneol substrate to cytochrome P450; electron flux and protein localization. CYP11A1, Adxp and Adrp are usually located in contact with inner mitochondrial membranes and are directed to the outside of the mitochondria by the removal of their respective mitochondrial targeting sequences. CYP11A1 then localizes to the plasma membrane but Adrp and Adxp are detected in the endoplasmic reticulum and cytosol as expected. The electron transfer chain that involves several subcellular compartments may control side chain cleavage activity in yeast. Interestingly, Tgl1p, a potential ester hydrolase, was found to enhance steroid productivity, probably through both the availability and/or the trafficking of the CYP11A1 substrate. Thus, the observation that the highest cellular levels of free ergosta-5-eneol are found in the plasma membrane suggests that the substrate is freely available for pregnenolone synthesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed2003

Computational methods for sequence mapping of large combinatorial libraries and deduced sequence signatures.

Here we describe a computational approach for the high-throughput sequence mapping of combinatorial libraries obtained by DNA shuffling. Original algorithms and their software implementation were developed for the automated and reliable analysis of hybridization data of differentially labeled oligonucleotide probes with PCR products spotted on DNA microarrays. This novel approach allows a context-dependent sequence attribution tolerant to fluctuations in experimental conditions and is well adapted to hybridization signals of variable qualities resulting from high-throughput PCR amplification from colonies. In addition, the analysis permits the calculation of sequence signatures that are characteristic of combinatorial library structure, defects, and diversity. The approach is of interest for the characterization and the equalization (library reduction to nonredundant structures) of combinatorial libraries involved in directed evolution and could be extrapolated to high-throughput polymorphism analysis.

Biotechnology↗
PubMed2002

Bioengineering and characterization of DNA-protein assemblies floating on supported membranes.

A biodevice involving thiolated ssDNA and engineered cytochrome b5 linked through a cis-platine bridge is described. This original nanostructure is associated to a supported membrane through a floating anchor, thus constituting a dynamic bidimensionnal DNA capture device contrasting with the constrained geometry of currently available DNA chips. Characterization by optical spectroscopy, surface plasmon resonance and gel electrophoresis demonstrated that an unique molecular supra-assembly featuring specific DNA recognition capability has been obtained. This device is characterized by the reversibility of its assembly, self-organization and fluidity properties and is of interest as a prototype to design new generations of DNA chip biosensors.

Base Sequence↗