NMR assignment and functional characterization of biotechnologically modified xanthan

Xanthan gum is one of the most important industrial microbial polysaccharides, yet comprehensive molecular-level structural characterization remains limited because of its high molecular weight, conformational rigidity, and severe spectral overlap in nuclear magnetic resonance spectroscopy. Here, we present a near-complete 1 H/ 13 C NMR chemical shift assignment of the xanthan repeating unit by combining multidimensional NMR spectroscopy with targeted glycosyltransferase knockout strains and controlled enzymatic depolymerization. Comparative analysis of wild-type xanthan and ΔgumI and ΔgumK variants enabled the unambiguous assignment of β-mannose and glucuronic acid resonances, and, by extension, of the remaining sugars, supporting future structural studies of engineered xanthan variants. In addition, we investigated how side-chain composition affects xanthan properties by analyzing genetically modified variants lacking specific acetyl, pyruvyl, or sugar residues. Temperature-dependent 1 H NMR determined order-disorder transition temperatures and revealed that removing pyruvate or negatively charged side-chain components increased thermal stability, whereas losing the terminal β-mannose destabilized the ordered conformation. Together, these results establish a robust NMR assignment for xanthan and provide new insights into how individual side-chain substituents influence the structural stability of this polysaccharide, facilitating the rational characterization of engineered xanthan variants and structurally closely related polysaccharides.