Expanding the chitin oligosaccharide portfolio by engineering NodC chitin synthases in Escherichia coli

Synthetic biology greatly accelerated the building process of potential microbial cell factories for the production of industrially relevant compounds, e.g., chitooligosaccharides (COS) which have an enormous application potential in multiple industries, i.e., pharma, cosmetics and agrifood. COS are produced by the heterologous expression of the chitin oligosaccharide synthase, NodC, in Escherichia coli, mainly yielding mixtures of chitintetraose (A4) and/or chitinpentaose (A5). We rationalised here product formation limitations based on molecular modelling of the structures of several NodC enzymes. We used this information to protein engineer NodC, rendering longer COS. Hence, an in vivo platform of defined COS-producing strains with different degrees of polymerisation was developed and experimentally characterised. Significantly, several strains were producing long COS, such as chitinhexaose (A6) and −heptaose (A7), not identified in any other natural producer. Additionally, other engineered strains efficiently produce almost 100% specific A4 or A5 product. Altogether, our results indicate that electrostatics-driven dynamics effects are to be considered in the molecular ruler hypothesis. Charge density at the transmembrane helices of NodC affects the opening of the integral binding pocket and in this way the length of the produced chitin oligomers can be modulated. As a result, the internal ruler mechanism elaborated and validated in this manuscript can serve as a guideline to perform site-directed mutagenesis at positions in related NodC and chitin synthase enzymes for both industrial applications as for identification of therapeutic targets.