Combinations of several enzymes in a production chain are preferred to “first generation” enzymatic processes (where the "single reaction - single enzyme" principle was followed), for the synthesis of compounds with high added value starting from simple and cheap substrates. An important requirement for obtaining control in "cascade enzymatic reactions" is the ability to deliver from one biocatalyst to the next one the various intermediates, limiting as much as possible the diffusion of the latter in the solvent. The spatial and specific organization of enzymes into artificial metabolons, through conventional immobilization methodologies, is however an open challenge. Our in vivo immobilization system, through the use of ASLtags, covalently binds two or three enzymes of interest, indirectly but with nanometric precision, allowing a modulation of the production chain activity without the need to purify the biocatalysts.
Our system allows the expression of enzymes of interest directly on the membrane of Gram(-) bacteria, through their gene fusion with innovative protein-tags (ASLtag), without complicated and expensive purification processes, considerably saving times and costs of the production chain. The ASLtag, in addition to exposing the enzyme to the solvent, covalently binds a part of its substrate, following its catalytic reaction. This property therefore allows the labelling of an enzyme fused to the ASLtag with a desired chemical group, if the latter is first conjugated to the substrate of the protein-tag. To date, there are three ASLtags with different substrate specificities: in the presence of linkers of different shapes and sizes conjugated to their substrates, the ASLtags bind covalently to each other, allowing a spatial arrangement with nanometric precision of enzymes on the surface of the bacterium, optimizing and/or modulating the activity of the "cascade enzymatic reactions".