Hélène Cordier

Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid

2,4-Dihydroxybutyric acid (DHB) is a molecule with considerable potential as a versatile chemical synthon. Notably, it may serve as a precursor for chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate, thus, targeting a considerable market in animal nutrition. However, no natural metabolic pathway exists for the biosynthesis of DHB. Here we have therefore conceived a three-step metabolic pathway for the synthesis of DHB starting from the natural metabolite malate. The pathway employs previously unreported malate kinase, malate semialdehyde dehydrogenase and malate semialdehyde reductase activities. The kinase and semialdehyde dehydrogenase activities were obtained by rational design based on structural and mechanistic knowledge of candidate enzymes acting on sterically cognate substrates. Malate semialdehyde reductase activity was identified from an initial screening of several natural enzymes, and was further improved by rational design. The pathway was expressed in a minimally engineered Escherichia coli strain and produces 1.8 g l−1 DHB with a molar yield of 0.15.

The PGM3 gene encodes the major phosphoribomutase in the yeast Saccharomyces cerevisiae

The phosphoglucomutases (PGM) Pgm1, Pgm2, and Pgm3 of the yeast Saccharomyces cerevisiae were tested for their ability to interconvert ribose‐1‐phosphate and ribose‐5‐phosphate. The purified proteins were studied in vitro with regard to their kinetic properties on glucose‐1‐phosphate and ribose‐1‐phosphate. All tested enzymes were active on both substrates with Pgm1 exhibiting only residual activity on ribose‐1‐phosphate. The Pgm2 and Pgm3 proteins had almost equal kinetic properties on ribose‐1‐phosphate, but Pgm2 had a 2000 times higher preference for glucose‐1‐phosphate when compared to Pgm3. The in vivo function of the PGMs was characterized by monitoring ribose‐1‐phosphate kinetics following a perturbation of the purine nucleotide balance. Only mutants with a deletion of PGM3 hyper‐accumulated ribose‐1‐phosphate. We conclude that Pgm3 functions as the major phosphoribomutase in vivo.

From EGEE Operations Portal towards EGI Operations Portal

EGEE to EGI structure based on NGIs evolution induces a large move from the operations that will rely on a sustainable and largely decentralized model. One of the key evolutions for the challenge in the regionalisation relies in the scalability and the flexibility required regarding information source types and information handling. For 5 years, we have developed and maintained a standard-based component that allows us to address both theses issues. This open-source tool, named Lavoisier, has been a critical success factor for the operations dashboard, one of the Operations Portal main features. Indeed, it enables coherent efficient and reliable data handling which is customizable and scalable, as Lavoisier is an extensible service designed to provide a unified view of data collected from multiple heterogeneous data sources. Data views are represented and accessed as XML documents through standard languages such as XSLT, XPath. Moreover, scalability and reliability are enforced by a caching mechanism adaptable to specific data sources and use-cases. We will namely expose how the concept and the implementation enable clear roles separation between plug-in developer, service configuration administrator or end-user. Also, maintainability of the portal code has increased dramatically as the latter is now independent from the data sources technology or from the cache management policies. Finally, integration of data has recently been simplified as the service administrator proceeds now through web interfaces

Construction of a synthetic metabolic pathway for the production of 2,4-dihydroxybutyric acid from homoserine

2,4-dihydroxybutyrate (DHB) is a precursor for the chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate. Since no annotated metabolic pathway exists for its microbial production from sugar, we have conceived a two-step synthetic metabolic pathway which converts the natural amino acid homoserine to DHB. The pathway proceeds through the homoserine transaminase-catalyzed deamination of homoserine to obtain 2-oxo-4-hydroxybutyrate (OHB), and continues with the reduction of OHB to DHB, which is catalyzed by an OHB reductase enzyme. We identified homoserine transaminase and OHB reductase activity in several candidate enzymes which act on sterically cognate substrates, and improved OHB reductase activity of lactate dehydrogenase A of Lactococcus lactis by structure-based enzyme engineering. Fed-batch cultivation of a homoserine-overproducing Escherichia coli strain which expressed homoserine transaminase and OHB reductase enzymes resulted in the production of 5.3g/L DHB at a yield of 0.1g/g.