Corrélation entre la biosynthèse des lipopeptides et le métabolisme de leurs précurseurs chez Bacillus subtilis

Genetic and metabolic engineering approaches were implemented to increase the precursor availability required for the biosynthesis of a lipopeptide produced by Bacillus subtilis. A metabolic network of amino acids metabolism in B. subtilis was first formalized through biocomputing tools. Then gene knock-out prediction was made using abstract interpretation and constraint solving. Predictions were based on genes which are directly or indirectly involved in the regulation of the genes involved in the biosynthesis of amino acid residues or in central carbon metabolism pathways. A markerless gene deletion strategy (Pop-in Pop-out technique) was adopted to carry out multiple deletions in a single strain and avoid antibiotic limitation. It was observed that the deletion of these genes have varied impact on lipopeptide production quantitatively and qualitatively. This work establishes that the precursor limitation problem of lipopeptide biosynthesis can be overcome by using this integrative approach.Dans ces travaux des approches de génie génétique et métabolique ont été mises en œuvre pour augmenter la fourniture des précurseurs nécessaire à la biosynthèse d’un lipopeptide produit par Bacillus subtilis. Un réseau métabolique du métabolisme des acides aminés chez B. subtilis a d'abord été formalisé et modélisé par des outils bioinformatique. Puis, grâce à l’utilisation de la programmation par contrainte et à l’interprétation abstraite des interruptions de gènes ont été prédites dans ce réseau. Les prédictions se sont basées sur des gènes qui sont directement ou indirectement impliqués dans la régulation des gènes responsables de la biosynthèse de certains acides aminés ou dans le métabolisme central du carbone. Une stratégie de suppression de gènes sans l’utilisation de marqueur antibiotique (technique du Pop-in Pop-out) a été entreprise pour mener à bien ces multiples délétions dans une même souche et en évitant ainsi une limitation par les antibiotiques. Les résultats obtenus suite à la délétion de ces gènes ont montrés un impact différent sur la production du lipopeptide tant d’un point de vue quantitatif que qualitatif. Ce travail a établi que la limitation de la biosynthèse du lipopeptide par la fourniture en précurseur peut être surmontée en utilisant cette approche intégrée

Bacillus subtilis-based microbial cell factories

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Modeling leucine's metabolic pathway and knockout prediction improving the production of surfactin, a biosurfactant from Bacillus subtilis

International audienceA Bacillus subtilis mutant strain overexpressing surfactin biosynthetic genes was previously constructed. In order to further increase the production of this biosurfactant, our hypothesis is that the surfactin precursors, especially leucine, must be overproduced. We present a three step approach for leucine overproduction directed by methods from computational biology. Firstly, we develop a new algorithm for gene knockout prediction based on abstract interpretation, which applies to a recent modeling language for reaction networks with partial kinetic information. Secondly, we model the leucine metabolic pathway as a reaction network in this language, and apply the knockout prediction algorithm with the target of leucine overproduction. Out of the 21 reactions corresponding to potential gene knockouts, the prediction algorithm selects 12 reactions. Six knockouts were introduced in B. subtilis 168 derivatives strains to verify their effects on surfactin production. For all generated mutants, the specific surfactin production is increased from 1.6 to 20.9 fold during the exponential growth phase, depending on the medium composition. These results show the effectiveness of the knockout prediction approach based on formal models for metabolic reaction networks with partial kinetic information, and confirms our hypothesis that precursors supply is one of the main parameter to optimize surfactin overproduction

MOLECULAR STRATEGIES FOR ADAPTING BACILLUS CELL TO OVERPRODUCE SURFACTIN IN BIOFILM BASED PROCESSES

The scope of this work was to develop molecular strategies to get genetically engineered strains designed to overproduce surfactin in biofilm based bioprocesses. Surfactin is a well-known lipopeptide biosurfactant synthesized by the non-ribosomal pathway [1]. It is composed of a ring of seven amino acid residues connected to a β-hydroxylated fatty acid chain of different length and isomery. The peptide moiety contains four leucines. The limiting factors of surfactin synthesis were identified and solved step by step in view to overproduce this compound but also to facilitate its recovery in continuous bioprocesses. Bacillus subtilis 168 or BSB1 were used as the main microbial chassis for this optimization process. Four molecular/differentiation steps were specifically addressed, i.e. gene transcription, post-translational modification, non-ribosomal synthetase activity and biofilm formation. Gene transcription and post-translational modifications were challenged by promoter replacement and through the insertion of an efficient sfp gene from B.subtilis ATCC 21332 [2]. This later is responsible for post-translational modification of surfactin synthetase from apo-form to holo-form by the addition of a phosphopantetheinate group. Regarding the non-ribosomal synthetase activity the main limiting factor is the intracellular concentration of surfactin precursor. A hybrid modelling approach involving metabolic pathways and the main transcriptional regulation steps was thus implemented for designing genetic engineering strategies in order to increase these intracellular concentrations. This hybrid model was more specifically focused on the metabolic pathways related to branched chain amino acids (BCAA: isoleucine, leucine and valine) for predicting the effects of gene disruption on the intracellular concentration of leucine and on surfactin biosynthesis [3,4]. The last optimization step was focused on strain engineering for increasing the ability of cells to grow in biofilm-based bioprocesses [5]. To this end, a set of Bacillus filamentous strains with restored exopolysaccharide (EPS) synthesis was designed and further checked for surfactin overproduction

Genetic engineering of the branched fatty acid metabolic pathway of Bacillus subtilis for the overproduction of surfactin C14 isoform

International audienceSurfactin, a lipopeptide produced by Bacillus subtilis, is one of the most powerful biosurfactants known. This molecule consists of a cyclic heptapeptide linked to a β-hydroxy fatty acid chain. The isomery and the length of the fatty acid (FA) chain are responsible for the surfactin’s activities. In this study, the gene codY, which encode for the global transcriptional regulator and the gene lpdV, located in the bkd operon (lpdV, bkdAA, bkdAB and bkdB genes), which is responsible for the last step of the branched chain amino acid (BCAA) degradation in acyl- CoA were deleted. The influence of these deletions on the quantitative and qualitative surfactin production was analysed. The surfactin production was quantified by RP-HPLC and the surfactin isoforms were characterized using LC-MS-MS and GC-MS analysis. The results obtained in the mutants showed an enhancement of surfactin specific production by a factor of 5.8 for the codY mutant and 1.4 for lpdV mutant. Moreover qualitative analysis of the lpdV mutant reveals that it mainly produced surfactin C14 isoform (2 fold more than the wild type) with linear FA chain. Complete analysis of the extracellular metabolites using 1H quantitative NMR reveals a reduced production of acetoin in this mutant. This work demonstrates for the first time an original approach to overproduce specifically surfactin with C14 FA chain