Understanding bottlenecks in the microbial production of partially acetylated chitooligosaccharides

Abstract 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) Rh NodC 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 Rh NodC and CDAs co-localize, suggesting potential interactions that influence production efficiency. Additionally, CDA expression induces significant stress responses, including upregulation of ibp A and cpx P 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 Rh NodC-CDA interactions and stress mitigation strategies to optimize scalable in vivo paCOS production. Highlights ‒ Co-expression of rhizobial NodC and chitin deacetylases reduces chitooligosaccharide yield. ‒ Conversion of COS into paCOS remains incomplete upon co-expression. ‒ Upregulation of stress responses suggests protein misfolding and membrane stress. ‒ NodC and chitin deacetylases possibly co-localize and affect cellular localization. ‒ Chitin deacetylase expression causes cell elongation up to 30 micrometers. ‒ Further study needed on rhizobial NodC-chitin deacetylase and substrate interactions.