Synthesis of Plant-Inspired <i>O</i> -Acetylated Hemicellulose Structures in the Yeast <i>Yarrowia lipolytica</i>

Hemicelluloses are a group of plant cell wall polysaccharides characterized by their high structural complexity. These glycans are part of an intricate composite polymer network that contribute to the mechanical strength and flexibility of plant cell walls. Hemicellulose structural and functional diversity is further enhanced by the presence of chemical modifications, such as <i>O</i>-acetylation, altering the polysaccharide's physicochemical properties and the overall functionality. Plant-derived hemicellulose glycans hold great promise for a range of biotechnological applications in a bioeconomy including biomaterials and pharmaceuticals. Synthetic biology approaches have the potential to produce hemicellulose polymers in microbial factories replicating the biosynthetic pathways observed in plants. In this study, we successfully reconstructed in the yeast <i>Yarrowia lipolytica</i> the biosynthesis of two hemicellulose backbone structures i.e., β-glucomannan (GM) and β-glucan, by the expression of glycosyltransferases of diverse plant origins. Oligosaccharide mass profiling combined with compositional and glycosidic linkage analysis confirmed the production of hemicellulose structures analogous to those found in the original plant systems. Furthermore, the additional expression of plant hemicellulose-specific <i>O</i>-acetyltransferases resulted in the biosynthesis of <i>O</i>-acetylated GM and <i>O</i>-acetylated glucan polymers, expanding the repertoire of hemicellulose structures produced in this yeast. These findings demonstrate the feasibility of generating not only compositionally diverse plant-like hemicellulose backbone polymers in microbial systems, but also more structurally complex <i>O</i>-acetylated variants beyond what is found in nature. The use of <i>Y. lipolytica</i> as a biofactory for designer glycans expands the potential of microbial glycoengineering and provides a platform for sustainable production of functionalized polysaccharides with tailored physicochemical properties optimized for specific biotechnological applications.