(235g) Novel Cellulose-Chitin Copolymers From Metabolically Engineered Gluconacetobacter Xylinus

Natural polymers offer impressive options for tailoring critical material features by manipulating their biological sources. Many proteins (e.g., collagens, elastins, silks) have been extensively utilized as biomaterials both in their native forms as well as their engineered variants. Structurally more robust and fibrillar polysaccharides, including cellulose, chitin and chitosan, however, have remained largely unexplored as biomaterials for tissue engineering and other biomedical applications. In particular, cellulose, which is the most abundant natural polymer, has found only limited applications in the narrow context of membrane filtration devices. A major technical challenge is the high crystallinity of the polymer and consequent poor enzymatic degradability. Another challenge is poor solubility, which limits material processing options. To address these challenges, we have metabolically engineered the cellulose producing bacterium Gluconacetobacter xylinus to incorporate N-acetylglucosamine (GlcNac) into cellulose microfbrils during biosynthesis. The UDP-GlcNac biosynthetic operon from the fungus Candida albicans was imported into G. xylinus. Quantification of intracellular metabolites using tandem mass spectrometry and HPLC confirmed elevated levels of both GlcNac and UDP-GlcNac in the engineered strain compared to the wild-type. Wide angle X-ray diffraction and FTIR showed low crystallinity in the modified cellulose produced from the engineered cells (modified bacterial cellulose; MBC) when compared with cellulose from wild-type cells (bacterial cellulose; BC). The MBC with reduced crystallinity was subject to more rapid enzymatic hydrolysis than BC. Our results suggest that the incorporation of the heterologous UDP-GlcNac operon is an essential first step towards the production of chimeric cellulosic biopolymers with tailorable material properties. Prospectively, the insights gained into the degradation of the modified sugar polymers will also aid in devising metabolic engineering strategies for the growth of novel cellulosic biomass feedstock amenable to efficient hydrolysis and conversion into value-added products.