Snoeck, S.; Malik, H.; Demeester, W.; Duchi, D.; Lips, W.; Guidi, C.; De Mey, M.

Chitooligosaccharides (COS) are versatile biomolecules with applications across food, pharmaceutical, and cosmetic industries. Expanding the COS portfolio, particularly with partially acetylated COS (paCOS) of defined degree and pattern of acetylation, is essential to unlocking their full potential. This study investigates the co-expression of chitin deacetylases (CDAs) with a chitooligosaccharide synthase (CHS) RhNodC in E. coli for in vivo paCOS production. While this approach shows promise, it is hampered by reduced overall (pa)COS yields and incomplete conversion of fully acetylated COS to paCOS. Our findings reveal that RhNodC and CDAs co-localize, suggesting potential interactions that influence production efficiency. Additionally, CDA expression induces significant stress responses, including upregulation of ibpA and cpxP promoters linked to inclusion body formation and membrane stress, respectively. This is accompanied by pronounced cellular elongation, further indicating cellular distress. These bottlenecks highlight the need for deeper exploration of RhNodC-CDA interactions and stress mitigation strategies to optimize scalable in vivo paCOS production.