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.