Synthetic Modules for the Biotechnological Generation and Evaluation of Specific Chitosan Oligomers

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Stephan Kolkenbrock, Bruno M. Moerschbacher

Institute of Plant Biology and Biotechnology, Germany

Chitosan, the linear heteropolysaccharide of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) residues, is the only naturally occuring polycationic polymer. As such, it has a number of highly attractive physico-chemical properties and, through interaction with the mostly anionic components of living cells, interesting biological activities, e.g., antimicrobial, plant strengthening, and immuno-modulatory activities. Chitosans are defined by their degree of polymerisation (DP), degree of acetylation (DA) and their pattern of acetylation (PA). We found a strong influence of DA and a less pronounced influence of DP on all biological activities. But even when using chitosans with well-defined DP and DA, the results of some bioassays tend to be variable, leading to the hypothesis that the PA has a crucial influence on their bioactivities. However, PA is not only difficult to determine, but also difficult to generate. In the ChitoBioEngineering project, we now combine different chitin synthases which produce chitin oligomers with a defined DP, with very specific chitin deacetylases which not only define the DA but also the PA of the chitosan oligomers generated. Combinations of these enzymes already give us a broad range of fully defined chitosan oligomers in vitro, but also enable us to establish biotechnological processes with different combinations of chitin synthase and chitin deacetylase modules in vivo to directly produce the desired, well-defined (DP, DA, PA) chitosan oligomers. Furthermore, we have not only established a chitosan oligomer sequencing procedure with highly specific exo-acting enzymes, but also managed to develop a fully synthetic biosensor for the detection of chitosan oligomers in vitro and in vivo. This biosensor is based on the idea of fluorescence indicator proteins, which generate a Frster resonance energy transfer signal in dependence on the concentration of the analyte. Thus, the biobricks are now available to build and evaluate cell factories to produce monoclonal oligochitosans.