No abstract

Mesotoga strain PhosAc3 was the first mesophilic cultivated member of the order Thermotogales. This genus currently contain two described species, M. prima and M. infera. Strain PhosAc3, isolated from a Tunisian digestor treating phosphogypsum, is phylogenetically closely related to M. prima strain MesG1.Ag.4.2T. Strain PhosAc3 has a genome of 3.1 Mb with a G+C content of 45.2%. It contains 3,051 protein-coding genes of which 74.6% have their best reciprocal BLAST hit in the genome of the type species, strain MesG1.Ag.4.2T. For this reason we propose to assign strain PhosAc3 as a novel ecotype of the Mesotoga prima species. However, in contrast with the M. prima type strain, (i) it does not ferment sugars but uses them only in the presence of elemental sulfur as terminal electron acceptor, (ii) it produces only acetate and CO2 from sugars, whereas strain MesG1.Ag.4.2T produces acetate, butyrate, isobutyrate, isovalerate, 2-methyl-butyrate and (iii) sulfides are also end products of the elemental sulfur reduction in theses growth conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/1944-3277-10-12) contains supplementary material, which is available to authorized users.

-Protéus is a biotechnology company specializing in the discovery, engineering and production of enzymes for industrial applications, as well as in the development of innovative bioprocesses involving these enzymes. Protéus is a subsidiary of the PCAS Group, actor in fine chemicals and specialty products and producer of high-value complex molecules. Enzymes allow considering unique functionalizations that are difficult to achieve by conventional chemical means. Examples involving the screening of our ready to use toolbox of lipases, the engineering of the well-known lipase CalB and the specific modifications of lipids will be presented. Keywords: lipids / lipases / protein engineering / bioprocesses / biocatalysisRésumé -Développement d'enzymes pour des applications industrielles. Protéus est une société de biotechnologie spécialisée dans la découverte, l'ingénierie et la production d'enzymes pour des applications industrielles, ainsi que dans le développement de bioprocédés innovants mettant en oeuvre ces enzymes. Protéus est une filiale du groupe PCAS actif dans le développement, la fabrication et la commercialisation de produits de chimie fine et de spécialités. Les enzymes permettent de produire de nouvelles molécules dans un contexte de chimie verte (économie d'atomes, conditions douces de mise en oeuvre, amélioration de la sélectivité, réduction de la toxicité) et d'envisager des fonctionnalisations uniques qui sont difficiles à obtenir par des moyens chimiques classiques. Des exemples de réalisation seront présentés avec notamment le criblage de notre toolbox enzymatique, l'ingénierie de la lipase issue de Candida antarctica et la fonctionnalisation de liaisons CH non activées.

Screening of prokaryotic genomes in order to identify enzymes with a desired catalytic activity can be performed in vivo in bacterial cells. We propose a strategy of in vitro expression screening of large prokaryotic genomic libraries based on Escherichia coli cell-free transcription-translation systems. Because cell-based expression may be limited by poor yield or protein misfolding, cell-free expression systems may be advantageous in permitting a more comprehensive screen under conditions optimized for the desired enzyme activity. However, monocistronic messages with an improved leader initiation context are typically used for protein production in vitro. Here, we describe successful use of a Pseudoalteromonas genomic DNA library for in vitro expression of DNA fragments carrying multiple open reading frames (ORFs) in the context of their authentic translation initiation sites and regulatory regions. We show that ORFs located far from the 5' and 3' ends of polycistronic transcripts can be expressed at a sufficient level in an in vitro transcription-translation system in order to allow functional screening. We demonstrate the overall cell-free functional screen strategy with the successful selection of an esterase from Pseudoalteromonas.

Cell-free expression-based screening is sometimes more suitable than cell-based assays for enzyme discovery. The advantage of cell-free systems for expression of toxic, poorly expressed, or insoluble proteins has already been well documented. Cell-free methods can advantageously replace cell-based ones when screening has to be performed on cell lysates prepared from harvested cells, for instance, when dealing with protein-ligand interactions particularly when the latter is hydrophobic. From our experience, cell-free extracts efficient in both transcription and translation can be prepared from potentially any microorganism. Here we present a general method for preparation of cell-free extracts from prokaryotic and eukaryotic cells, selection of the best systems, and optimized conditions for specific protein expression. The method allows to select proteins for their ability to bind a selected target, to identify the inhibitors of such binding, or to identify novel enzymatic activities.