NMR assignment and functional characterization of biotechnologically modified xanthan

Abstract

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 1H/13C 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 xanthan from the wild-type strain and the{Delta} gumI and{Delta} gumK variants enabled unambiguous assignment of {beta}-mannose and glucuronic acid resonances and, by extension, 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 1H NMR determined order-disorder transition temperatures and revealed that removing pyruvate or negatively charged side-chain components increased thermal stability, whereas losing the terminal {beta}-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.

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