Host physiological constraints limit microbial production of partially acetylated chitooligosaccharides

Chitooligosaccharides (COS) are versatile biomolecules with applications across food, pharmaceutical, and cosmetic industries. Expanding the COS portfolio, particularly with partially acetylated COS (paCOS) possessing defined structural characteristics, including degree of deacetylation and pattern of acetylation, is essential for advancing structure-function studies and enabling tailored applications. This study investigates the heterologous co-expression of chitin deacetylases (CDAs) with a chitin oligosaccharide synthase (oCS) RhNodC in Escherichia coli for the in vivo production of paCOS. Although this approach enables paCOS formation, co-expression of NodC and CDAs results in reduced COS yields and incomplete conversion of fully acetylated COS into paCOS. To identify potential cellular bottlenecks underlying these limitations, stress responses were monitored using a stress promoter-based fluorescent reporter system. CDA co-expression activated protein quality control and membrane-associated stress responses. Furthermore, confocal microscopy revealed altered intracellular localization of both RhNodC and CDAs compared to individual expression, with enzymes detected in the cytoplasm, membrane and inclusion bodies. Co-expression also resulted in pronounced cellular elongation, further indicating cellular stress during pathway implementation. Together, these findings reveal cellular bottlenecks associated with enzyme co-expression and highlight the need for deeper exploration of RhNodC-CDA co-expression and approaches to mitigate cellular stress to improve in vivo paCOS production.